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Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell–like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas. Significance: Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1–BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma.

Supplementary Table from BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma

Supplementary Figure from BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma

Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell–like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas.Significance:Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1–BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma.See related commentary by Beytagh and Weiss, p. 2730.See related article by Mo et al., p. 2906.This article is highlighted in the In This Issue feature, p. 2711

Inhibition of immune checkpoints, including cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and its ligand PD-L1, has demonstrated exciting and durable remissions across a spectrum of malignancies. Combinatorial regimens blocking complementary immune checkpoints further enhance the therapeutic benefit. The activity of these agents for patients with glioblastoma, a generally lethal primary brain tumor associated with significant systemic and microenvironmental immunosuppression, is not known. We therefore systematically evaluated the antitumor efficacy of murine antibodies targeting a broad panel of immune checkpoint molecules, including CTLA-4, PD-1, PD-L1, and PD-L2 when administered as single-agent therapy and in combinatorial regimens against an orthotopic, immunocompetent murine glioblastoma model. In these experiments, we observed long-term tumor-free survival following single-agent anti–PD-1, anti–PD-L1, or anti–CTLA-4 therapy in 50%, 20%, and 15% of treated animals, respectively. Combination therapy of anti–CTLA-4 plus anti–PD-1 cured 75% of the animals, even against advanced, later-stage tumors. In long-term survivors, tumor growth was not seen upon intracranial tumor rechallenge, suggesting that tumor-specific immune memory responses were generated. Inhibitory immune checkpoint blockade quantitatively increased activated CD8+ and natural killer cells and decreased suppressive immune cells in the tumor microenvironment and draining cervical lymph nodes. Our results support prioritizing the clinical evaluation of PD-1, PD-L1, and CTLA-4 single-agent targeted therapy as well as combination therapy of CTLA-4 plus PD-1 blockade for patients with glioblastoma. Cancer Immunol Res; 4(2); 124–35. ©2015 AACR.

NUT carcinoma (NC), characterized most commonly by the BRD4-NUTM1 fusion, is a rare, aggressive variant of squamous carcinoma with no effective treatment. BRD4-NUT drives growth and maintains the poorly differentiated state of NC by activating pro-growth genes such as MYC, through the formation of massive, hyperacetylated, superenhancer-like domains termed megadomains. BRD4-NUT–mediated hyperacetylation of chromatin is facilitated by the chromatin-targeting tandem bromodomains of BRD4, combined with NUT, which recruits the histone acetyltransferase, p300. Here, we developed a high-throughput small-molecule screen to identify inhibitors of transcriptional activation by NUT. In this dCAS9-based GFP-reporter assay, the strongest hits were diverse histone deacetylase (HDAC) inhibitors. Two structurally unrelated HDAC inhibitors, panobinostat and the novel compound, IRBM6, both repressed growth and induced differentiation of NC cells in proportion to their inhibition of NUT transcriptional activity. These two compounds repressed transcription of megadomain-associated oncogenic genes, such as MYC and SOX2, while upregulating pro-differentiation, non-megadomain–associated genes, including JUN, FOS, and key cell-cycle regulators, such as CDKN1A. The transcriptional changes correlate with depletion of BRD4-NUT from megadomains, and redistribution of the p300/CBP-associated chromatin acetylation mark, H3K27ac, away from megadomains toward regular enhancer regions previously populated by H3K27ac. In NC xenograft models, we demonstrated that suppression of tumor growth by panobinostat was comparable with that of bromodomain inhibition, and when combined they improved both survival and growth suppression.Implications:The findings provide mechanistic and preclinical rationale for the use of HDAC inhibitors, alone or combined with other agents, in the treatment of NUT carcinoma.

Supplementary Table 1

Supplementary Figures 1-6 and Supplementary Methods

Supplementary Table 6

Supplementary Methods

The extraordinary activity of high-dose cyclophosphamide against some high-grade lymphomas was described nearly 60 years ago. Here we address mechanisms that mediate cyclophosphamide activity in bona fide human double-hit lymphoma. We show that antibody resistance within the bone marrow (BM) is not present upon early engraftment but develops during lymphoma progression. This resistance required a high tumor:macrophage ratio, was recapitulated in spleen by partial macrophage depletion, and was overcome by multiple, high-dose alkylating agents. Cyclophosphamide induced endoplasmic reticulum (ER) stress in BM-resident lymphoma cells in vivo that resulted in ATF4-mediated paracrine secretion of VEGFA, massive macrophage infiltration, and clearance of alemtuzumab-opsonized cells. BM macrophages isolated after cyclophosphamide treatment had increased phagocytic capacity that was reversed by VEGFA blockade or SYK inhibition. Single-cell RNA sequencing of these macrophages identified a “super-phagocytic” subset that expressed CD36/FCGR4. Together, these findings define a novel mechanism through which high-dose alkylating agents promote macrophage-dependent lymphoma clearance.Significance:mAbs are effective against only a small subset of cancers. Herein, we recapitulate compartment-specific antibody resistance and define an ER stress–dependent mechanism induced by high-dose alkylating agents that promotes phagocytosis of opsonized tumor cells. This approach induces synergistic effects with mAbs and merits testing across additional tumor types.See related commentary by Duval and De Palma, p. 834.This article is highlighted in the In This Issue feature, p. 813

Supplementary Methods

Supplemental Table

Supplementary Table 3

Supplementary Table 2

Supplementary Figure Legends 1-4

Supplementary Table 4

Figure S1. Rationale and readout for the dCAS9-NUT chemical screen. Figure S2. IRBM6 is highly active in vitro and in vivo. Figure S3. IRBM6 induces transcriptional programs leading to growth arrest and differentiation in NC. Figure S4. IRBM6 treatment induces redistribution of BRD4-NUT and H3K27ac.

Supplemental Figure 1. Bioluminescence plots of individual animals following treatment with immune checkpoint blocking mAbs. Supplemental Figure 2. Immune cell infiltrates within intracranial GL261-luc2 tumor following treatment with isotype control IgG or combination therapy with CTLA-4 plus PD-1 mAb therapy. Supplemental Figure 3: Treatment antibody and detection antibody for PD-1 do not share same the epitope. Supplemental Figure 4. Representative immunohistochemistry results of intratumoral CD3+, CD4+ and CD8+ T cell infiltrates following immune checkpoint therapy with mAbs against CTLA-4, PD-1 and both CTLA-4 and PD-1. Supplemental Figure 5. Immune cell infiltration within the draining cervical lymph node (cLN) of GL261-luc2 tumor-bearing mice following treatment with isotype control IgG or combination therapy with mAbs against CTLA-4 plus PD-1. Supplemental Figure 6. Comparisons of chemokine expression levels in sera of mice treated with checkpoint blockade. Supplemental Figure 7: Expression of checkpoint ligands and receptors on GL261-Luc cells in vitro. Supplementary Table 1. Arrangement of chemokines on the Mouse Chemokine Antibody Array.

Supplementary Table 5

Supplementary Figure Legends

LISA analysis of master regulators of differentially expressed genes

Supplementary Data including supplementary methods, supplementary figures, and supplementary figure legends.

GSEA Hallmarks analysis of RNA-seq of bulk lung tumors

RNA-seq data of bulk lung tumors from sgControl RPP, sgNotch1 RPP, sgNotch2 RPP, and sgAscl1 RPP CRISPR-based SCLC GEMMs

RNA-seq data of the SCLC adherent cells that are either Notch-WT or Notch-Mutant

Neuroendocrine to nonneuroendocrine plasticity supports small cell lung cancer (SCLC) tumorigenesis and promotes immunogenicity. Approximately 20% to 25% of SCLCs harbor loss-of-function (LOF) NOTCH mutations. Previous studies demonstrated that NOTCH functions as a SCLC tumor suppressor, but can also drive nonneuroendocrine plasticity to support SCLC growth. Given the dual functionality of NOTCH, it is not understood why SCLCs select for LOF NOTCH mutations and how these mutations affect SCLC tumorigenesis. In a CRISPR-based genetically engineered mouse model of SCLC, genetic loss of Notch1 or Notch2 modestly accelerated SCLC tumorigenesis. Interestingly, Notch-mutant SCLCs still formed nonneuroendocrine subpopulations, and these Notch-independent, nonneuroendocrine subpopulations were driven by Runx2-mediated regulation of Rest. Notch2-mutant nonneuroendocrine cells highly express innate immune signaling genes including stimulator of interferon genes (STING) and were sensitive to STING agonists. This work identifies a Notch-independent mechanism to promote nonneuroendocrine plasticity and suggests that therapeutic approaches to activate STING could be selectively beneficial for SCLCs with NOTCH2 mutations.Significance:A genetically engineered mouse model of NOTCH-mutant SCLC reveals that nonneuroendocrine plasticity persists in the absence of NOTCH, driven by a RUNX2-REST–dependent pathway and innate immune signaling.

Neuroendocrine to nonneuroendocrine plasticity supports small cell lung cancer (SCLC) tumorigenesis and promotes immunogenicity. Approximately 20% to 25% of SCLCs harbor loss-of-function (LOF) NOTCH mutations. Previous studies demonstrated that NOTCH functions as a SCLC tumor suppressor, but can also drive nonneuroendocrine plasticity to support SCLC growth. Given the dual functionality of NOTCH, it is not understood why SCLCs select for LOF NOTCH mutations and how these mutations affect SCLC tumorigenesis. In a CRISPR-based genetically engineered mouse model of SCLC, genetic loss of Notch1 or Notch2 modestly accelerated SCLC tumorigenesis. Interestingly, Notch-mutant SCLCs still formed nonneuroendocrine subpopulations, and these Notch-independent, nonneuroendocrine subpopulations were driven by Runx2-mediated regulation of Rest. Notch2-mutant nonneuroendocrine cells highly express innate immune signaling genes including stimulator of interferon genes (STING) and were sensitive to STING agonists. This work identifies a Notch-independent mechanism to promote nonneuroendocrine plasticity and suggests that therapeutic approaches to activate STING could be selectively beneficial for SCLCs with NOTCH2 mutations. Significance: A genetically engineered mouse model of NOTCH-mutant SCLC reveals that nonneuroendocrine plasticity persists in the absence of NOTCH, driven by a RUNX2-REST–dependent pathway and innate immune signaling.

How cancer cells adapt to evade the therapeutic effects of drugs targeting oncogenic drivers is poorly understood. Here we report an epigenetic mechanism leading to the adaptive resistance of triple-negative breast cancer (TNBC) to fibroblast growth factor receptor (FGFR) inhibitors. Prolonged FGFR inhibition suppresses the function of BRG1-dependent chromatin remodelling, leading to an epigenetic state that derepresses YAP-associated enhancers. These chromatin changes induce the expression of several amino acid transporters, resulting in increased intracellular levels of specific amino acids that reactivate mTORC1. Consistent with this mechanism, addition of mTORC1 or YAP inhibitors to FGFR blockade synergistically attenuated the growth of TNBC patient-derived xenograft models. Collectively, these findings reveal a feedback loop involving an epigenetic state transition and metabolic reprogramming that leads to adaptive therapeutic resistance and provides potential therapeutic strategies to overcome this mechanism of resistance. Li et al. define an adaptive resistance mechanism against FGFR inhibitor treatment in breast cancer attributed to loss of BRG1 chromatin recruitment, reactivation of YAP-dependent enhancers and upregulation of amino acid-induced mTORC1 activity.

NUT carcinoma (NC), characterized most commonly by the BRD4-NUTM1 fusion, is a rare, aggressive variant of squamous carcinoma with no effective treatment. BRD4-NUT drives growth and maintains the poorly differentiated state of NC by activating pro-growth genes such as MYC, through the formation of massive, hyperacetylated, superenhancer-like domains termed megadomains. BRD4-NUT–mediated hyperacetylation of chromatin is facilitated by the chromatin-targeting tandem bromodomains of BRD4, combined with NUT, which recruits the histone acetyltransferase, p300. Here, we developed a high-throughput small-molecule screen to identify inhibitors of transcriptional activation by NUT. In this dCAS9-based GFP-reporter assay, the strongest hits were diverse histone deacetylase (HDAC) inhibitors. Two structurally unrelated HDAC inhibitors, panobinostat and the novel compound, IRBM6, both repressed growth and induced differentiation of NC cells in proportion to their inhibition of NUT transcriptional activity. These two compounds repressed transcription of megadomain-associated oncogenic genes, such as MYC and SOX2, while upregulating pro-differentiation, non-megadomain–associated genes, including JUN, FOS, and key cell-cycle regulators, such as CDKN1A. The transcriptional changes correlate with depletion of BRD4-NUT from megadomains, and redistribution of the p300/CBP-associated chromatin acetylation mark, H3K27ac, away from megadomains toward regular enhancer regions previously populated by H3K27ac. In NC xenograft models, we demonstrated that suppression of tumor growth by panobinostat was comparable with that of bromodomain inhibition, and when combined they improved both survival and growth suppression. Implications: The findings provide mechanistic and preclinical rationale for the use of HDAC inhibitors, alone or combined with other agents, in the treatment of NUT carcinoma.

High-grade gliomas (HGG) afflict both children and adults and respond poorly to current therapies. Epigenetic regulators have a role in gliomagenesis, but a broad, functional investigation of the impact and role of specific epigenetic targets has not been undertaken. Using a two-step, in vitro/in vivo epigenomic shRNA inhibition screen, we determine the chromatin remodeler BPTF to be a key regulator of adult HGG growth. We then demonstrate that BPTF knockdown decreases HGG growth in multiple pediatric HGG models as well. BPTF appears to regulate tumor growth through cell self-renewal maintenance, and BPTF knockdown leads these glial tumors toward more neuronal characteristics. BPTF’s impact on growth is mediated through positive effects on expression of MYC and MYC pathway targets. HDAC inhibitors synergize with BPTF knockdown against HGG growth. BPTF inhibition is a promising strategy to combat HGG through epigenetic regulation of the MYC oncogenic pathway.

SUMMARYDiffuse midline glioma (DMG) is a uniformly fatal pediatric cancer driven by oncohistones that do not readily lend themselves to drug development. To identify druggable targets for DMG, we conducted a genome-wide CRISPR screen that reveals a DMG selective dependency on the de novo pathway for pyrimidine biosynthesis. This metabolic vulnerability reflects an elevated rate of uridine/uracil degradation that depletes DMG cells of substrates for the alternate salvage pathway for pyrimidine biosynthesis. A clinical stage inhibitor of DHODH (rate limiting enzyme in the de novo pathway) diminishes UMP pools, generates DNA damage, and induces apoptosis through suppression of replication forks--an “on target” effect, as shown by uridine rescue. MALDI mass spectroscopy imaging demonstrates that this DHODH inhibitor (BAY2402234) accumulates in brain at therapeutically relevant concentrations, suppresses de novo pyrimidine biosynthesis in vivo, and prolongs survival of mice bearing intracranial DMG xenografts, highlighting BAY2402234 as a promising therapy against DMGs.

The extraordinary activity of high-dose cyclophosphamide against some high-grade lymphomas was described nearly 60 years ago. Here we address mechanisms that mediate cyclophosphamide activity in bona fide human double-hit lymphoma. We show that antibody resistance within the bone marrow (BM) is not present upon early engraftment but develops during lymphoma progression. This resistance required a high tumor:macrophage ratio, was recapitulated in spleen by partial macrophage depletion, and was overcome by multiple, high-dose alkylating agents. Cyclophosphamide induced endoplasmic reticulum (ER) stress in BM-resident lymphoma cells in vivo that resulted in ATF4-mediated paracrine secretion of VEGFA, massive macrophage infiltration, and clearance of alemtuzumab-opsonized cells. BM macrophages isolated after cyclophosphamide treatment had increased phagocytic capacity that was reversed by VEGFA blockade or SYK inhibition. Single-cell RNA sequencing of these macrophages identified a “super-phagocytic” subset that expressed CD36/FCGR4. Together, these findings define a novel mechanism through which high-dose alkylating agents promote macrophage-dependent lymphoma clearance. Significance: mAbs are effective against only a small subset of cancers. Herein, we recapitulate compartment-specific antibody resistance and define an ER stress–dependent mechanism induced by high-dose alkylating agents that promotes phagocytosis of opsonized tumor cells. This approach induces synergistic effects with mAbs and merits testing across additional tumor types. See related commentary by Duval and De Palma, p. 834. This article is highlighted in the In This Issue feature, p. 813

The interleukin-3 receptor α subunit, CD123, is expressed in many hematologic malignancies including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Tagraxofusp (SL-401) is a CD123-targeted therapy consisting of interleukin-3 fused to a truncated diphtheria toxin payload. Factors influencing response to tagraxofusp other than CD123 expression are largely unknown. We interrogated tagraxofusp resistance in patients and experimental models and found that it was not associated with CD123 loss. Rather, resistant AML and BPDCN cells frequently acquired deficiencies in the diphthamide synthesis pathway, impairing tagraxofusp's ability to ADP-ribosylate cellular targets. Expression of DPH1, encoding a diphthamide pathway enzyme, was reduced by DNA CpG methylation in resistant cells. Treatment with the DNA methyltransferase inhibitor azacitidine restored DPH1 expression and tagraxofusp sensitivity. We also developed a drug-dependent ADP-ribosylation assay in primary cells that correlated with tagraxofusp activity and may represent an additional novel biomarker. As predicted by these results and our observation that resistance also increased mitochondrial apoptotic priming, we found that the combination of tagraxofusp and azacitidine was effective in patient-derived xenografts treated in vivo. These data have important implications for clinical use of tagraxofusp and led to a phase 1 study combining tagraxofusp and azacitidine in myeloid malignancies.

Blockade of immune cell co-inhibitory receptor PD-1 using monoclonal antibodies (mAbs) allows the development of anti-tumor immunity in various solid tumors and lymphoid malignancies. We recently demonstrated that PD-1 blockade elicits an anti-tumor immune response resulting in tumor rejection and long-term survival in approximately 50% of mice with intra-cranial GL-261 glioblastoma, despite the absence of accumulating CD8+ cytotoxic T cells in the tumor or draining lymph nodes. In this investigation, we provide evidence for the role of conventional CD4+ T cells and the innate immune response in PD-1 mediated anti-glioma immunity in this model. In response to anti-PD-1 monotherapy, intratumoral CD4+ T cells, but not CD8+ T cells, expressed significantly elevated levels of IFN-γ and TNF-α pro-inflammatory cytokines and the cytotoxic enzyme, granzyme B. Tbet, GATA3, and EOMES, transcription factors required for T cell proliferation, activation, and effector function, were also up-regulated in CD4+, but not CD8+ T cells in the brains of mice treated with PD-1 mAbs when compared to controls. We demonstrated that depletion of CD4+ or CD8+ T cells, but not NK, was sufficient to completely ablate anti-PD-1-mediated tumor eradication and long-term survival. CD4+ T cell activation was accompanied by the classical activation and M1 polarization of resident microglia and tumor-infiltrating macrophages. Together, these studies demonstrate for the first time a role for CD4+ T cells and the innate immune response in the eradication of glioblastoma by PD-1 blockade.

Targeted inhibitors of essential oncogenic kinases induce high rates of clinical response but cure few patients due to the persistence of minimal residual disease (MRD). BCR-ABL mutant leukemias are a classic example of this paradigm where patients usually achieve deep remissions followed by near inevitable relapses. Multiple factors have been shown to influence how an individual patient’s leukemic cells will navigate treatment including differentiation state, mutational background, and communication with the microenvironment. Here, we use BCR-ABL-rearranged acute lymphoblastic leukemia (BCR-ABL ALL) to interrogate cell-autonomous features leading to therapeutic resistance using low-input single-cell assays. Specifically, we use a combination of primary samples and PDX models to dissect aberrant developmental hierarchies and monitor leukemic cell transcriptional and biophysical phenotype at pretreatment, MRD, and relapse. Using machine learning, we relate malignant B cells to normal development, allowing us to define leukemic developmental programs and demonstrate that these have consequences for the time to progression as well as the genetic alterations seen at relapse. Further, we determine that there are unique biophysical features tied to leukemic developmental states and that these integrative properties co-evolve with transcriptional state over the course of treatment. Finally, we demonstrate in PDX studies that it may be possible to intercept relapse by targeting specific features of MRD cells. Together, these data suggest that significant developmental hierarchies exist in ALL, tumor subpopulations can be identified directly within MRD, and their phenotypic and molecular characterization can be exploited to therapeutic effect. Citation Format: Peter S. Winter, Andrew Navia, Haley Strouf, Mahnoor Mirza, Jennyfer Galvez-Reyes, Nolawit Mulugeta, Laura Bilal, Nezha Senhaji, Peter Dennis, Catharine S. Leahy, Kay Shigemori, Foster Powers, Alejandro Gupta, Nicholas Calistri, Alex Van Scoyk, Kristen Jones, Huiyun Liu, Kristen E. Stevenson, Robert Kimmerling, Mark Stevens, David M. Weinstock, Scott R. Manalis, Mark A. Murakami, Alex K. Shalek. Aberrant leukemic developmental hierarchies and MRD-specific targeting informed by single-cell biophysical and molecular profiling [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA20.

The original version of this Article omitted the following from the Acknowledgements: This work was supported by the Luke's Army Pediatric Cancer Research Fund St. Baldrick's Scholar Award. This has now been corrected in both the PDF and HTML versions of the Article.

Blockade of immune cell co-inhibitory receptor PD-1 using monoclonal antibodies (mAbs) allows the development of anti-tumor immunity in various solid tumors and lymphoid malignancies. We recently demonstrated that PD-1 blockade elicits an anti-tumor immune response resulting in tumor rejection and long-term survival in approximately 50% of mice with intra-cranial GL-261 glioblastoma, despite the absence of accumulating CD8+ cytotoxic T cells in the tumor or draining lymph nodes. In this investigation, we provide evidence for the role of conventional CD4+ T cells and the innate immune response in PD-1 mediated anti-glioma immunity in this model. In response to anti-PD-1 monotherapy, intratumoral CD4+ T cells, but not CD8+ T cells, expressed significantly elevated levels of IFN-γ and TNF-α pro-inflammatory cytokines and the cytotoxic enzyme, granzyme B. Tbet, GATA3, and EOMES, transcription factors required for T cell proliferation, activation, and effector function, were also up-regulated in CD4+, but not CD8+ T cells in the brains of mice treated with PD-1 mAbs when compared to controls. We demonstrated that depletion of CD4+ or CD8+ T cells, but not NK, was sufficient to completely ablate anti-PD-1-mediated tumor eradication and long-term survival. CD4+ T cell activation was accompanied by the classical activation and M1 polarization of resident microglia and tumor-infiltrating macrophages. Moreover, CSF-1 inhibition with PLX3397 to deplete microglia and macrophages did not affect tumor growth, but did lead to a significant survival advantage when combined with anti-PD-1 mAb. Together, these studies demonstrate for the first time a role for CD4+ T cells and the innate immune response in the eradication of glioblastoma by PD-1 blockade.

Background. Glioblastoma (GBM) is poorly responsive to current chemotherapy. The nuclear transporter exportin 1 (XPO1, CRM1) is often highly expressed in GBM, which may portend a poor prognosis. Here, we determine the efficacy of novel selective inhibitors of nuclear export (SINE) specific to XPO1 in preclinical models of GBM. Methods. Seven patient-derived GBM lines were treated with 3 SINE compounds (KPT-251, KPT-276, and Selinexor) in neurosphere culture conditions. KPT-276 and Selinexor were also evaluated in a murine orthotopic patient-derived xenograft (PDX) model of GBM. Cell cycle effects were assayed by flow cytometry in vitro and immunohistochemistry in vivo. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase 3/7 activity assays. Results. Treatment of GBM neurosphere cultures with KPT-276, Selinexor, and KPT-251 revealed dose-responsive growth inhibition in all 7 GBM lines [range of half-maximal inhibitory concentration (IC50), 6‐354 nM]. In an orthotopic PDX model, treatment with KPT-276 and Selinexor demonstrated pharmacodynamic efficacy, significantly suppressed tumor growth, and prolonged animal survival. Cellular proliferation was not altered with SINE treatment. Instead, induction of apoptosis was apparent both in vitro and in vivo with SINE treatment, without overt evidence of neurotoxicity. Conclusions. SINE compounds show preclinical efficacy utilizing in vitro and in vivo models of GBM, with induction of apoptosis as the mechanism of action. Selinexor is now in early clinical trials in solid and hematological malignancies. Based on these preclinical data and excellent brain penetration, we have initiated clinical trials of Selinexor in patients with relapsed GBM.

Background Magnetic Resonance Imaging (MRI) relies on optimal scanning parameters to achieve maximal signal-to-noise ratio (SNR) and high contrast-to-noise ratio (CNR) between tissues resulting in high quality images. The optimization of such parameters is often laborious, time consuming, and user-dependent, making harmonization of imaging parameters a difficult task. In this report, we aim to develop and validate a computer simulation technique that can reliably provide "optimal in vivo scanning parameters" ready to be used for in vivo evaluation of disease models. Methods A glioblastoma murine model was investigated using several MRI imaging methods. Such MRI methods underwent a simulated and an in vivo scanning parameter optimization in pre- and post-contrast conditions that involved the investigation of tumor, brain parenchyma and cerebrospinal fluid (CSF) CNR values in addition to the time relaxation values of the related tissues. The CNR tissues information were analyzed and the derived scanning parameters compared in order to validate the simulated methodology as a reliable technique for "optimal in vivo scanning parameters" estimation. Results The CNRs and the related scanning parameters were better correlated when spin-echo-based sequences were used rather than the gradient-echo-based sequences due to augmented inhomogeneity artifacts affecting the latter methods. "Optimal in vivo scanning parameters" were generated successfully by the simulations after initial scanning parameter adjustments that conformed to some of the parameters derived from the in vivo experiment. Conclusion Scanning parameter optimization using the computer simulation was shown to be a valid surrogate to the in vivo approach in a glioblastoma murine model yielding in a better delineation and differentiation of the tumor from the contralateral hemisphere. In addition to drastically reducing the time invested in choosing optimal scanning parameters when compared to an in vivo approach, this simulation program could also be used to harmonize MRI acquisition parameters across scanners from different vendors.

Inhibition of immune checkpoints, including cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and its ligand PD-L1, has demonstrated exciting and durable remissions across a spectrum of malignancies. Combinatorial regimens blocking complementary immune checkpoints further enhance the therapeutic benefit. The activity of these agents for patients with glioblastoma, a generally lethal primary brain tumor associated with significant systemic and microenvironmental immunosuppression, is not known. We therefore systematically evaluated the antitumor efficacy of murine antibodies targeting a broad panel of immune checkpoint molecules, including CTLA-4, PD-1, PD-L1, and PD-L2 when administered as single-agent therapy and in combinatorial regimens against an orthotopic, immunocompetent murine glioblastoma model. In these experiments, we observed long-term tumor-free survival following single-agent anti–PD-1, anti–PD-L1, or anti–CTLA-4 therapy in 50%, 20%, and 15% of treated animals, respectively. Combination therapy of anti–CTLA-4 plus anti–PD-1 cured 75% of the animals, even against advanced, later-stage tumors. In long-term survivors, tumor growth was not seen upon intracranial tumor rechallenge, suggesting that tumor-specific immune memory responses were generated. Inhibitory immune checkpoint blockade quantitatively increased activated CD8+ and natural killer cells and decreased suppressive immune cells in the tumor microenvironment and draining cervical lymph nodes. Our results support prioritizing the clinical evaluation of PD-1, PD-L1, and CTLA-4 single-agent targeted therapy as well as combination therapy of CTLA-4 plus PD-1 blockade for patients with glioblastoma. Cancer Immunol Res; 4(2); 124–35. ©2015 AACR.

Purpose: To determine if epacadostat, an oral indoleamine 2,3-dioxygenase (IDO1) inhibitor has therapeutic benefit against GBM when administered as single agent and with PD-1 blocking antibody. Methods: An initial survival experiment was performed to assess efficacy and was followed by an identical repeat experiment for validation. 1X105 luciferized GL261 cells, a murine GBM tumor line derived from intracerebral methylcholanthrene implantation, were stereotactically implanted intracranially in albino syngeneic C57BL/6 mice. Mice with increasing bioluminescence on days 3 and 6 were randomized (n=8/group) to receive treatment beginning on day 6: anti-PD-1 (332.8H3, mouse IgG1; 500 μg intraperitoneal (IP) on day 6, 250 μg q 3 days X 7); epacadostat (Incyte Corporation, orally dosed at 300 mg/kg/day for 5 days on/2days off for 3 weeks); anti-PD-1 + epacadostat; and control therapy (isotype IgG antibody IP and 0.5% methocel in water). Tumor response assessments were performed by quantifying bioluminescence and survival. A re-challenge experiment was performed in long-term survivors to assess for tumor immune responses capable of preventing relapse. All long-term surviving mice (defined as ≥ 100 days) from the efficacy experiment were injected with 1X105 GL261 cells in the contralateral hemisphere and followed for survival. Results: In both preclinical efficacy experiments, median survival in the epacadostat monotherapy group did not differ from controls (approximately 30 days). Four of 8 mice (50%) treated with anti-PD-1 were long-term survivors in both efficacy experiments. In the epacadostat plus anti-PD-1 combination group, 81% of the mice were long-term survivors (7 of 8 in experiment 1 and 6 of 8 in experiment 2). Of note, none of the long-term surviving mice developed evidence of tumor; thus the median survival among the anti-PD-1 and epacadostat plus anti-PD-1 combination groups were both > 100 days. In the re-challenge study, all of the mice who underwent GL261 re-inoculation survived > 100 more days with no evidence of tumor recurrence. Conclusions: IDO1 inhibition with epacadostat increased the eradication rate of anti-PD-1 therapy in an orthotopic syngeneic GBM model and long term survivors rejected tumor following orthotopic re-challenge. Further combinatorial studies incorporating IDO inhibitor therapy for GBM, including mechanistic studies, are warranted. Citation Format: David A. Reardon, Prafulla C. Gokhale, Sarah R. Klein, Kristen L. Jones, Paul T. Kirschmeier, Maria Speranza, Holly Koblish, Peggy Scherle, Lance Leopold, Robert Newton, Gordon J. Freeman. Inhibition of IDO1 with epacadostat enhances anti-tumor efficacy of PD-1 blockade in a syngeneic glioblastoma (GBM) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 572. doi:10.1158/1538-7445.AM2017-572

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) have been discovered in several cancer types and cause the neurometabolic syndrome D2-hydroxyglutaric aciduria (D2HGA). The mutant enzymes exhibit neomorphic activity resulting in production of D2-hydroxyglutaric acid (D-2HG). To study the pathophysiological consequences of the accumulation of D-2HG, we generated transgenic mice with conditionally activated IDH2R140Q and IDH2R172K alleles. Global induction of mutant IDH2 expression in adults resulted in dilated cardiomyopathy, white matter abnormalities throughout the central nervous system (CNS), and muscular dystrophy. Embryonic activation of mutant IDH2 resulted in more pronounced phenotypes, including runting, hydrocephalus, and shortened life span, recapitulating the abnormalities observed in D2HGA patients. The diseased hearts exhibited mitochondrial damage and glycogen accumulation with a concordant up-regulation of genes involved in glycogen biosynthesis. Notably, mild cardiac hypertrophy was also observed in nude mice implanted with IDH2R140Q-expressing xenografts, suggesting that 2HG may potentially act in a paracrine fashion. Finally, we show that silencing of IDH2R140Q in mice with an inducible transgene restores heart function by lowering 2HG levels. Together, these findings indicate that inhibitors of mutant IDH2 may be beneficial in the treatment of D2HGA and suggest that 2HG produced by IDH mutant tumors has the potential to provoke a paraneoplastic condition.

BACKGROUND: Outcome for glioblastoma (GBM) remains dismal and innovative treatment strategies are desperately needed. Growing data support the contribution of local and systemic immune checkpoint molecules mediating immunosuppression by GBM tumors. METHODS: Luciferized GL261 cells (GL261-Luc) were stereotactically implanted intracranially in albino C57BL/6 mice. Cohorts of mice with growing tumors (increasing bioluminescence) were treated with murine monoclonal antibodies (MAb) against PD-1, PD-L1, PD-L2, and CTLA-4 (single agent and combinations) every 3 days X 8 beginning 6 days following tumor implantation and were followed for overall survival. Subsets of mice underwent MRI as well as analysis of tumor infiltrating immune cells, and systemic immune cells and immunocytokines. Tumor re-challenge experiments were performed among long-term surviving mice. RESULTS: Immune checkpoint blockade was well tolerated with no apparent morbidity or mortality among treated animals. Improved survival was noted among mice treated with immune checkpoint blockade compared to appropriate controls. The most robust survival benefit was noted among mice treated with combinatorial therapy including 12/18 (75%) mice treated with anti-PD-1 + anti-CTLA-4 MAbs alive at 140 days with no evidence of residual tumor. MRI imaging among long-term surviving animals demonstrated clear evidence of initial tumor growth followed by regression and eradication of tumors. Long-term surviving mice appeared fully intact neurologically and exhibited no evidence of tumor growth following re-challenge of GL261-Luc cells injected subcutaneously. Characterization of tumor infiltrating immune cells as well as systemic immune cells and immunocytokines is ongoing. CONCLUSION: Immune checkpoint blockade provides significant survival benefit and appears safe in this immunocompetent, orthotopic syngeneic GBM model. Re-challenge experiments demonstrate evidence of long-term immunologic memory and further elucidation of underlying mechanisms of immune-mediated anti-tumor activity is ongoing. These data strongly support the evaluation of immune checkpoint inhibitors among patients with GBM.

2084 Background: Outcome for glioblastoma (GBM) remains dismal and innovative treatment strategies are desperately needed. Growing data support the contribution of local and systemic immune checkpoint molecules mediating immunosuppression by GBM tumors. Methods: Luciferized GL261 cells (GL261-Luc) were stereotactically implanted intracranially in albino C57BL/6 mice. Cohorts of mice with growing tumors (increasing bioluminescence) were treated with murine monoclonal antibodies (MAb) against PD-1, PD-L1, PD-L2, and CTLA-4 (single agent and combinations) every 3 days X 8 beginning 6 days following tumor implantation and were followed for overall survival. Subsets of mice underwent MRI as well as analysis of tumor infiltrating immune cells, and systemic immune cells and immunocytokines. Tumor re-challenge experiments were performed among long-term surviving mice. Results: Immune checkpoint blockade was well tolerated with no apparent morbidity or mortality among treated animals. Improved survival was noted a...

With the advent of genome sequence information, in addition to capabilities for cloning and expressing genes of interest in foreign hosts, a wide range of hyperthermophilic enzymes have become accessible for potential applications for biocatalytic processes. Not only can these enzymes be useful for strategic opportunities at high temperatures, but there may also be advantages that derive from their relatively low activity at suboptimal temperatures. Examples of several possible ways in which hyperthermophilic enzymes could be used are presented, including cases where they could serve as environmentally benign alternatives in existing industrial processes.