Commentary: preclinical efficacy of immune-checkpoint monotherapy does not recapitulate corresponding biomarkers-based clinical predictions in glioblastoma by Garg et al. (2017).

Preclinical modeling and gene expression analyses have yielded distinct observations for the role of immune checkpoint, IDO1, in glioblastoma (GBM). Accordingly, our recent work differs with Garg et al. (2017) with respect to IDO1 among preclinical and bioinformatic GBM datasets. Here, we discuss the methodological differences that affected study interpretation, and potentially, future clinical decision-making for IDO1-targeting approaches against GBM.

IDO1 Inhibition Synergizes with Radiation and PD-1 Blockade to Durably Increase Survival Against Advanced Glioblastoma.

Purpose: Glioblastoma is the most aggressive primary brain tumor in adults with a median survival of 15-20 months. Numerous approaches and novel therapeutics for treating glioblastoma have been investigated in the setting of phase III clinical trials, including a recent analysis of the immune checkpoint inhibitor, nivolumab (anti-PD-1), which failed to improve recurrent glioblastoma patient survival. However, rather than abandoning immune checkpoint inhibitor treatment for glioblastoma, which has shown promise in other types of cancer, ongoing studies are currently evaluating this therapeutic class when combined with other agents.Experimental Design: Here, we investigated immunocompetent orthotopic mouse models of glioblastoma treated with the potent CNS-penetrating IDO1 enzyme inhibitor, BGB-5777, combined with anti-PD1 mAb, as well as radiotherapy, based on our recent observation that tumor-infiltrating T cells directly increase immunosuppressive IDO1 levels in human glioblastoma, the previously described reinvigoration of immune cell functions after PD-1 blockade, as well as the proinflammatory effects of radiation.Results: Our results demonstrate a durable survival benefit from this novel three-agent treatment, but not for any single- or dual-agent combination. Unexpectedly, treatment efficacy required IDO1 enzyme inhibition in non-glioblastoma cells, rather than tumor cells. Timing of effector T-cell infiltration, animal subject age, and usage of systemic chemotherapy, all directly impacted therapy-mediated survival benefit.Conclusions: These data highlight a novel and clinically relevant immunotherapeutic approach with associated mechanistic considerations that have formed the basis of a newly initiated phase I/II trial for glioblastoma patients. Clin Cancer Res; 24(11); 2559-73. ©2018 AACR.

Impact of immunotherapy among patients with melanoma brain metastases managed with radiotherapy.

BACKGROUND: Patients with melanoma brain metastases (MBM) have been excluded from trials evaluating immunotherapy in melanoma. As such, immunotherapy's role in MBM is poorly understood, particularly in combination with radiotherapy. METHODS: The National Cancer Database was queried for patients with MBM receiving brain radiotherapy. They were classified according to immunotherapy receipt. Multivariate Cox regression was performed to identify factors associated with survival. RESULTS: Among 1287 patients, 185 received immunotherapy. Factors associated with improved survival included younger age, academic facility, lower extracranial disease burden, stereotactic radiotherapy, chemotherapy, and immunotherapy. CONCLUSIONS: Adding immunotherapy to radiotherapy for MBM is associated with improved survival.

Infiltrating T Cells Increase IDO1 Expression in Glioblastoma and Contribute to Decreased Patient Survival.

Purpose: Indoleamine 2,3 dioxygenase 1 (IDO1) mediates potent immunosuppression in multiple preclinical models of cancer. However, the basis for elevated IDO1 expression in human cancer, including the most common primary malignant brain tumor in adults, glioblastoma (GBM), is poorly understood. The major objective of this study is to address this gap in our understanding of how IDO1 expression contributes to the biology of GBM, and whether its level of expression is a determinant of GBM patient outcome.Experimental Design: Patient-resected GBM, The Cancer Genome Atlas, human T-cell:GBM cocultures, as well as nu/nu, NOD-scid, and humanized (NSG-SGM3-BLT) mice-engrafted human GBM form the basis of our investigation.Results:In situ hybridization for IDO1 revealed transcript expression throughout patient-resected GBM, whereas immunohistochemical IDO1 positivity was highly variable. Multivariate statistical analysis revealed that higher levels of IDO1 transcript predict a poor patient prognosis (P = 0.0076). GBM IDO1 mRNA levels positively correlated with increased gene expression for markers of cytolytic and regulatory T cells, in addition to decreased patient survival. Humanized mice intracranially engrafted human GBM revealed an IFNγ-associated T-cell-mediated increase of intratumoral IDO1Conclusions: Our data demonstrate that high intratumoral IDO1 mRNA levels correlate with a poor GBM patient prognosis. It also confirms the positive correlation between increased GBM IDO1 levels and human-infiltrating T cells. Collectively, this study suggests that future efforts aimed at increasing T-cell-mediated effects against GBM should consider combinatorial approaches that coinhibit potential T-cell-mediated IDO1 enhancement during therapy. Clin Cancer Res; 23(21); 6650-60. ©2017 AACR.

The Boosting Potential of Bacteria in Cancer Immunotherapy.

Salmonella Typhimurium, engineered to express flagellin B, recently demonstrated unprecedented tumor control through a TLR-dependent mechanism. Here, we review new observations that support the potential of utilizing modified bacteria to enhance antitumor immunity. We also discuss the implications of these findings for clinical applications, including immune checkpoint blockade therapies.

Non-tumor cell IDO1 predominantly contributes to enzyme activity and response to CTLA-4/PD-L1 inhibition in mouse glioblastoma.

Glioblastoma (GBM) is the most common malignant brain tumor in adults with a median survival of 14.6months. A contributing factor to GBM aggressiveness is the intratumoral expression of the potently immunosuppressive enzyme, indoleamine 2,3 dioxygenase 1 (IDO1). The enzymatic activity of IDO1 is associated with the conversion of tryptophan into downstream kynurenine (Kyn), which has previously been hypothesized to contribute toward the suppression of tumor immunity. Utilizing the syngeneic, immunocompetent, intracranial GL261 cell GBM model, we previously demonstrated that tumor cell, but not non-tumor cell IDO1, suppresses T cell-mediated brain tumor regression in mice. Paradoxically, we also showed that the survival advantage mediated by immune checkpoint blockade is abrogated by non-tumor cell IDO1 deficiency. Here, we have built on our past observations and confirm the maladaptive role of tumor cell IDO1 in a novel mouse GBM model. We also demonstrate that, non-tumor cells, rather than mouse GBM cells, are the dominant contributor to IDO1-mediated enzyme activity. Finally, we show the novel associations between maximally-effective immune-checkpoint blockade-mediated survival, non-tumor cell IDO1 and intra-GBM Kyn levels. These data suggest for the first time that, GBM cell-mediated immunosuppression is IDO1 enzyme independent, while the survival benefits of immune checkpoint blockade require non-tumor cell IDO1 enzyme activity. Given that current clinical inhibitors vary in their mechanism of action, in terms of targeting IDO1 enzyme activity versus enzyme-independent effects, this work suggests that choosing an appropriate IDO1 pharmacologic will maximize the effectiveness of future immune checkpoint blockade approaches.

Improving vaccine efficacy against malignant glioma.

The effective treatment of adult and pediatric malignant glioma is a significant clinical challenge. In adults, glioblastoma (GBM) accounts for the majority of malignant glioma diagnoses with a median survival of 14.6 mo. In children, malignant glioma accounts for 20% of primary CNS tumors with a median survival of less than 1 y. Here, we discuss vaccine treatment for children diagnosed with malignant glioma, through targeting EphA2, IL-13Rα2 and/or histone H3 K27M, while in adults, treatments with RINTEGA, Prophage Series G-100 and dendritic cells are explored. We conclude by proposing new strategies that are built on current vaccine technologies and improved upon with novel combinatorial approaches.

Tumor relapse prevented by combining adoptive T cell therapy with Salmonella typhimurium.

We recently reported that therapeutic vaccination with live tumor antigen-producing Salmonella typhimurium rescues dysfunctional endogenous T cell responses and eradicates long-established tumors refractory to αCTLA-4 and αPD-L1 checkpoint inhibitor blockade. Here, we show that live intravenously injected or heat-killed (HK) intratumorally injected Salmonella typhimurium, even when not producing tumor antigen, synergize with adoptive T cell therapy to eradicate tumors. These data demonstrate that the combination of adoptive T cell transfer with the injection of live or dead Salmonella typhimurium is a promising approach for cancer treatment.

Immunotherapy for cancer in the central nervous system: Current and future directions.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and still remains incurable. Although immunotherapeutic vaccination against GBM has demonstrated immune-stimulating activity with some promising survival benefits, tumor relapse is common, highlighting the need for additional and/or combinatorial approaches. Recently, antibodies targeting immune checkpoints were demonstrated to generate impressive clinical responses against advanced melanoma and other malignancies, in addition to showing potential for enhancing vaccination and radiotherapy (RT). Here, we summarize the current knowledge of central nervous system (CNS) immunosuppression, evaluate past and current immunotherapeutic trials and discuss promising future immunotherapeutic directions to treat CNS-localized malignancies.

Molecular Pathways: Targeting IDO1 and Other Tryptophan Dioxygenases for Cancer Immunotherapy.

Indoleamine 2, 3-dioxygenase 1 (IDO1), IDO2, and tryptophan 2, 3-dioxygenase (TDO) comprise a family of enzymes that catalyze the first- and rate-limiting step associated with the catabolic conversion of tryptophan (Trp) into kynurenine (Kyn). Through subsequent enzymatic and spontaneous reactions, Kyn is further converted into the energetic substrates, NAD(+) and ATP, to fuel cellular metabolic functions. Coincidently, the depletion of Trp and accumulation of Kyn has been demonstrated to induce effector T-cell apoptosis/dysfunction and immunosuppressive regulatory T-cell induction, respectively. Similar to other immune checkpoints, IDO1 and TDO are suggested to be important targets for immunotherapeutic intervention. This is represented by the recent growth of efforts to inhibit the Trp-to-Kyn pathway as a means to control immunosuppression. Inhibitors currently in clinical trials, INCB024360, GDC-0919, indoximod, and an IDO1 peptide-based vaccine, are being evaluated for their efficacy against a wide range of cancers including melanoma, glioblastoma, non-small cell lung, pancreatic, and/or breast cancer, as well as metastatic disease. Despite the rapid development of potent clinical grade inhibitors, strategic questions remain. Here, we review the state of the literature with respect to current therapeutic inhibitors of tryptophan catabolism, evaluation of those efforts preclinically and clinically, compensatory changes that occur with therapeutic targeting, as well as newly recognized signaling features that raise critical questions to the field. Given the rapidly evolving interest in determining how IDO1/TDO, and to an unknown extent, IDO2, can be targeted for increasing cancer immunotherapeutic efficacy, we present a brief but comprehensive analysis that addresses critical questions, while highlighting the mechanics that remain to be explored.

IV Administered Gadodiamide Enters the Lumen of the Prostatic Glands: X-Ray Fluorescence Microscopy Examination of a Mouse Model.

OBJECTIVE: Dynamic contrast-enhanced MRI (DCE-MRI) has become a standard component of multiparametric protocols for MRI examination of the prostate, and its use is incorporated into current guidelines for prostate MRI examination. Analysis of DCE-MRI data for the prostate is usually based on the distribution of gadolinium-based agents, such as gadodiamide, into two well-mixed compartments, and it assumes that gadodiamide does not enter into the glandular lumen. However, this assumption has not been directly tested. The purpose of this study was to use x-ray fluorescence microscopy (XFM) imaging in situ to measure the concentration of gadodiamide in the epithelia and lumens of the prostate of healthy mice after IV injection of the contrast agent. MATERIALS AND METHODS: Six C57Bl6 male mice (age, 28 weeks) were sacrificed 10 minutes after IV injection of gadodiamide (0.13 mmol/kg), and three mice were sacrificed after saline injection. Prostate tissue samples obtained from each mouse were harvested and frozen; 7-µm-thick slices were sectioned for XFM imaging, and adjacent 5-µm-thick slices were sectioned for H and E staining. Elemental concentrations were determined from XFM images. RESULTS: A mean (± SD) baseline concentration of gadolinium of 0.01 ± 0.01 mM was determined from XFM measurements of prostatic tissue samples when no gadodiamide was administered, and it was used to determine the measurement error. When gadodiamide was added, the mean concentrations of gadolinium in the epithelia and lumens in 32 prostatic glands from six mice were 1.00 ± 0.13 and 0.36 ± 0.09 mM, respectively. CONCLUSION: Our data suggest that IV administration of gadodiamide results in uptake of contrast agent by the glandular lumens of the mouse prostate. We were able to quantitatively determine gadodiamide distributions in mouse prostatic epithelia and lumens.

Radiotherapy and immune checkpoint blockade: potential interactions and future directions.

Combining radiation and immune checkpoint blockade provides synergistic antitumor responses in animal models and a small subset of patients. New preclinical data have provided mechanistic insight into this treatment interaction and identification of therapeutic targets to optimize this approach.

Adoptively transferred immune T cells eradicate established tumors despite cancer-induced immune suppression.

Myeloid-derived CD11b(+)Gr1(+) suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are considered a major obstacle for effective adoptive T cell therapy. Myeloid cells suppress naive T cell proliferation ex vivo and can prevent the generation of T cell responses in vivo. We find, however, that adoptively transferred immune T cells eradicate well-established tumors in the presence of MDSCs and TAMs, which are strongly immunosuppressive ex vivo. These MDSCs and TAMs were comparable in numbers and immunosuppressive capacity among different tumor models. Longitudinal microscopy of tumors in vivo revealed that after T cell transfer, tumor vasculature and cancer cells disappeared simultaneously. During T cell-mediated tumor destruction, the tumor stroma contained abundant myeloid cells (mainly TAMs) that retained their suppressive properties. Preimmunized but not naive mice resisted immune suppression caused by an unrelated tumor burden, supporting the idea that in vivo, myeloid immunosuppressive cells can suppress naive but not memory T cell responses.

Relapse or eradication of cancer is predicted by peptide-major histocompatibility complex affinity.

Cancers often relapse after adoptive therapy, even though specific T cells kill cells from the same cancer efficiently in vitro. We found that tumor eradication by T cells required high affinities of the targeted peptides for major histocompatibility complex (MHC) class I. Affinities of at least 10 nM were required for relapse-free regression. Only high-affinity peptide-MHC interactions led to efficient cross-presentation of antigen, thereby stimulating cognate T cells to secrete cytokines. These findings highlight the importance of targeting peptides with high affinity for MHC class I when designing T cell-based immunotherapy.

High-affinity peptide-based anticancer vaccination to overcome resistance to immunostimulatory antibodies.

We tested how to eradicate long-established immunogenic tumors that were resistant to the monoclonal antibody-mediated blockade of PD-L1 (PD-1 ligand 1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4). Bacterial vaccination with a tumor-specific peptide exhibiting a high affinity for its respective MHC molecule consistently eradicated tumors when combined with a PD-L1 blocking antibody. This approach can be translated to the clinic by combining cancer cell whole-exome sequencing with algorithms to identify mutant peptides with high peptide-MHC binding affinity.

Antigen-specific bacterial vaccine combined with anti-PD-L1 rescues dysfunctional endogenous T cells to reject long-established cancer.

Immunogenic tumors grow progressively even when heavily infiltrated by CD8(+) T cells. We investigated how to rescue CD8(+) T cell function in long-established immunogenic melanomas that contained a high percentage of endogenous PD-1(+) tumor-specific CD8(+) T cells that were dysfunctional. Treatment with αPD-L1 and αCTLA-4 blocking antibodies did not prevent tumors from progressing rapidly. We then tested exogenous tumor-specific antigen delivery into tumors using Salmonella Typhimurium A1-R to increase antigen levels and generate a proinflammatory tumor microenvironment. Antigen-producing A1-R rescued the endogenous tumor-specific CD8(+) T cell response: proliferation was induced in the lymphoid organs and effector function was recovered in the tumor. Treatment with antigen-producing A1-R led to improved mouse survival and resulted in 32% rejection of long-established immunogenic melanomas. Following treatment with antigen-producing A1-R, the majority of tumor-specific CD8(+) T cells still expressed a high level of PD-1 in the tumor. Combining antigen-producing A1-R with αPD-L1 blocking antibody enhanced the expansion of tumor-specific CD8(+) T cells and resulted in 80% tumor rejection. Collectively, these data demonstrate a powerful new therapeutic approach to rescue dysfunctional endogenous tumor-specific CD8(+) T cells and eradicate advanced immunogenic tumors.

Densely granulated murine NK cells eradicate large solid tumors.

Natural killer (NK) cells inhibit early stages of tumor formation, recurrence, and metastasis. Here, we show that NK cells can also eradicate large solid tumors. Eradication depended on the massive infiltration of proliferating NK cells due to interleukin 15 (IL-15) released and presented by the cancer cells in the tumor microenvironment. Infiltrating NK cells had the striking morphologic feature of being densely loaded with periodic acid-Schiff-positive, diastase-resistant granules, resembling uterine NK cells. Perforin-mediated killing by these densely granulated NK cells was essential for tumor eradication. Expression of the IL-15 receptor α on cancer cells was needed to efficiently induce granulated NK cells, and expression on host stromal cells was essential to prevent tumor relapse after near complete destruction. These results indicate that IL-15 released at the cancer site induces highly activated NK cells that lead to eradication of large solid tumors.

Cytosolic group IVa phospholipase A2 mediates IL-8/CXCL8-induced transmigration of human polymorphonuclear leukocytes in vitro.

BACKGROUND: Cytosolic gIVaPLA2 is a critical enzyme in the generation of arachidonate metabolites and in induction of beta2-integrin adhesion in granulocytes. We hypothesized that gIVaPLA2 activation also is an essential downstream step for post adhesive migration of PMN in vitro. METHODS: Migration of PMNs caused by IL-8/CXCL8 was assessed using a transwell migration chamber. PMNs were pretreated with two structurally unrelated inhibitors of gIVaPLA2, arachidonyl trifluoromethylketone (TFMK) or pyrrophenone, prior to IL-8/CXCL8 exposure. The fraction of migrated PMNs present in the lower chamber was measured as total myeloperoxidase content. GIVaPLA2 enzyme activity was analyzed using [14C-PAPC] as specific substrate F-actin polymerization and cell structure were examined after rhodamine-phalloidin staining. RESULTS: IL-8/CXCL8-induced migration of PMNs was elicited in concentration- and time-dependent manner. Time-related phosphorylation and translocation of cytosolic gIVaPLA2 to the nucleus was observed for PMNs stimulated with IL-8/CXCL8 in concentration sufficient to cause upstream phosphorylation of MAPKs (ERK-1/2 and p38) and Akt/PKB. Inhibition of gIVaPLA2 corresponded to the magnitude of blockade of PMN migration. Neither AA nor LTB4 secretion was elicited following IL-8/CXCL8 activation. In unstimulated PMNs, F-actin was located diffusely in the cytosol; however, a clear polarized morphology with F-actin-rich ruffles around the edges of the cell was observed after activation with IL-8/CXCL8. Inhibition of gIVaPLA2 blocked change in cell shape and migration caused by IL-8/CXCL8 but did not cause F-actin polymerization or translocation of cytosolic F-actin to inner leaflet of the PMN membrane. CONCLUSION: We demonstrate that IL-8/CXCL8 causes a) phosphorylation and translocation of cytosolic gIVaPLA2 to the nucleus, b) change in cell shape, c) polymerization of F-actin, and d) chemoattractant/migration of PMN in vitro. Inhibition of gIVaPLA2 blocks the deformability and subsequent migration of PMNs caused by IL-8/CXCL8. Our data suggest that activation of gIVaPLA2 is an essential step in PMN migration in vitro.