Erik Ladomersky

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.

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 IDO1. Conclusions: 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.

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.

Improving vaccine efficacy against malignant glioma

ABSTRACT 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.

Advanced age negatively impacts survival in an experimental brain tumor model

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, with an average age of 64 years at the time of diagnosis. To study GBM, a number of mouse brain tumor models have been utilized. In these animal models, subjects tend to range from 6 to 12 weeks of age, which is analogous to that of a human teenager. Here, we examined the impact of age on host immunity and the gene expression associated with immune evasion in immunocompetent mice engrafted with syngeneic intracranial GL261. The data indicate that, in mice with brain tumors, youth conveys an advantage to survival. While age did not affect the tumor-infiltrating T cell phenotype or quantity, we discovered that old mice express higher levels of the immunoevasion enzyme, IDO1, which was decreased by the presence of brain tumor. Interestingly, other genes associated with promoting immunosuppression including CTLA-4, PD-L1 and FoxP3, were unaffected by age. These data highlight the possibility that IDO1 contributes to faster GBM outgrowth with advanced age, providing rationale for future investigation into immunotherapeutic targeting in the future.

IDO1 in cancer: a Gemini of immune checkpoints

Indoleamine 2, 3-dioxygenase 1 (IDO1) is a rate-limiting metabolic enzyme that converts the essential amino acid tryptophan (Trp) into downstream catabolites known as kynurenines. Coincidently, numerous studies have demonstrated that IDO1 is highly expressed in multiple types of human cancer. Preclinical studies have further introduced an interesting paradox: while single-agent treatment with IDO1 enzyme inhibitor has a negligible effect on decreasing the established cancer burden, approaches combining select therapies with IDO1 blockade tend to yield a synergistic benefit against tumor growth and/or animal subject survival. Given the high expression of IDO1 among multiple cancer types along with the lack of monotherapeutic efficacy, these data suggest that there is a more complex mechanism of action than previously appreciated. Similar to the dual faces of the astrological Gemini, we highlight the multiple roles of IDO1 and review its canonical association with IDO1-dependent tryptophan metabolism, as well as documented evidence confirming the dispensability of enzyme activity for its immunosuppressive effects. The gene transcript levels for IDO1 highlight its strong association with T-cell infiltration, but the lack of a universal prognostic significance among all cancer subtypes. Finally, ongoing clinical trials are discussed with consideration of IDO1-targeting strategies that enhance the efficacy of immunotherapy for cancer patients.

The Kynurenine/Tryptophan Ratio and Glioblastoma Patients Treated with Hsppc-96 Vaccine

The discovery that immunotherapy is a clinically-relevant approach for the treatment of malignant tumors is revolutionizing patient care. In adults diagnosed with glioblastoma (GBM), an aggressive and incurable primary brain tumor, autologous HSPPC-96 vaccination provides a significant increase in overall survival. However, all GBM patients eventually succumb to their disease, providing rationale for discovering new methods that proactively identify individuals that will respond, optimally. Of the immunosuppressive mediators that contribute to the inhibition of productive tumor immunity, indoleamine 2,3 dioxygenase 1 (IDO1), a rate-limiting enzyme that catabolizes tryptophan (Trp) into kynurenine (Kyn), has been demonstrated to be expressed at elevated levels in patients with malignant glioma. Recently, our group determined that a correlation exists between peripheral blood Trp and Kyn levels in GBM patients and the association with overall survival after HSPPC-96 treatment. Our findings indicate that the Kyn/Trp ratio may be a useful benchmark for identifying GBM patients with a higher likelihood to survive longer after vaccination. The relevance to future clinical trials, the limitations of brain tumor models to address these findings and the role of IDO1 versus tryptophan dioxygenase (TDO) in the maintenance of peripheral Trp and Kyn levels, is discussed.

Abstract A064: IDO1 expression stratifies glioblastoma patient survival and correlates with dominantly immunosuppressive pathways

Purpose : The objective of this study was to evaluate the prognostic significance of the immunosuppressive molecule indoleamine 2, 3-dioxygenase ( IDO1 ) mRNA expression in patients with low-grade glioma (LGG) and glioblastoma (GBM). Experimental Design : Hi-RNA-seq. Illumina data mined from the cancer genome atlas (TCGA) reflecting 475 LGG (WHO grade II and III) and 172 GBM patients were utilized to characterize the pattern of IDO1 mRNA expression. Kaplan-Meier (KM) survival analysis and multivariate Cox proportional hazards regression were conducted to evaluate the significance of IDO1 expression as a stratification marker in glioma patients. mRNA expression of proinflammatory and immunosuppressive factor genes were also investigated for their correlation with IDO1 mRNA expression. Ivy GAP analysis was further utilized to localize IDO1 mRNA within different GBM anatomic niches. Results: Expression profiling of the TCGA revealed a distinct pattern of progressively increasing IDO1 mRNA levels correlating with decreased survival both in LGG and GBM patients. In grade III LGG and GBM, IDO1 mRNA expression was significantly decreased when mutant isocitrate dehydrogenase 1 and 2 (m IDH1/2 ) was coincidently expressed when compared to samples with wild-type IDH1/2 expression (mean difference = 1.51, P P IDO1 mRNA expression levels to be an independent prognostic factor for survival among LGG ( P = 0.0115; HR = 1.68) and GBM ( P = 0.0076; HR = 1.82) patients. Pearson9s correlation analysis identified significant associations among GBM between IDO 1 and the genes encoding immunosuppressive factors including PD-L1 ( r = 0.2993, P PD-L2 ( r = 0.4871, P PD-1 ( r = 0.3416, P CTLA-4 ( r = 0.3534, P STAT3 ( r = 0.2366, P = 0.0018), CD39 ( r = 0.2691, P = 0.0004), BTLA ( r = 0.2981, P Lag3 ( r = 0.2567, P = 0.0007), FoxP3 ( r = 0.1865, P FGL2 ( r = 0.4267, P IDO1 mRNA levels were higher in the cellular (1.398 ± 0.1257, mean ± SEM) and necrotic (1.543 ± 0.1489) GBM zones, when compared to infiltrating (0.9303 ± 0.0786) and leading margins (0.9064 ± 0.1224). Additionally, for the IDO1 paralog genes IDO2 and TDO2 , which encodes indoleamine 2, 3-dioxygenase 2 and tryptophan dioxygenase respectively, no overall differential expression pattern were observed between different pathological grades nor a significant stratification in LGG and GBM patient survival. Conclusions : Our data show for the first time that IDO1 mRNA levels can be used as an independent prognostic variable for patients with LGG and/or GBM. Given that ongoing IDO1-targeted immunotherapy clinical trials aim to inhibit enzymatic activity in malignant glioma, our data suggest that targeted subject enrollment may result in a better clinical response to therapy. Citation Format: Lijie Zhai, Matthew Genet, Erik Ladomersky, Kristen Lauing, Meijing Wu, David Binder, Leo Kim, Jeremy Rich, Craig Horbinski, C. David James, Jeffrey A. Sosman, Orin Bloch, Derek A. Wainwright. IDO1 expression stratifies glioblastoma patient survival and correlates with dominantly immunosuppressive pathways [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A064.

Abstract B018: A novel IDO1 inhibitor combined with targeted immunotherapy durably increases survival in a mouse model of glioblastoma

Glioblastoma (GBM) is the most common primary brain tumor, accounting for 54% of malignant glioma diagnoses. Over the course of the past 30 years, a diagnosis of GBM has remained fatal even after maximum surgical resection, radiotherapy (RT), and chemotherapy, with a median overall survival of 14.6 months. The immunosuppressive microenvironment of GBM is a major contributor to the poor patient outcome. Expression of IDO1, as well as the accumulation of tumor-infiltrating regulatory T cells contribute to the avoidance of immune surveillance. Although current immunotherapies have had some success in extending patient survival, combinatorial treatment approaches addressing both tumor growth and the potent immunosuppression may prove to be more effective. This work aimed to determine the efficacy of a novel, pharmaceutical-grade, blood brain barrier-penetrating small molecule IDO1 inhibitor, BGB-5777, in combination with PD-1 blockade and/or whole brain radiation in an immunocompetent mouse GBM model. METHODS/RESULTS: All mice were intracranially-engrafted 2×105 GL261 (syngeneic to B6 background) cells to recapitulate brain tumors. At 14 days post-intracranial injection (dp-ic.), mice were treated with IgG alone as a control (n = 7), 2Gy RT for 5 days (n = 8), 500 ug (loading dose), followed by three 200 ug maintenance doses given every 3 days, of PD-1 mAb (J43) (n = 8), or 100mg/kg BGB-5777 for 4 weeks (n = 10), with a median overall survival (OS) of 25, 25, 32, and 26.5 days, respectively. Mice treated with dual therapies including RT and PD-1 mAb (n = 10), RT and BGB-5777 (n = 8), or PD-1 mAb and BGB-5777 (n = 9), had a median OS of 30, 39, and 32 days, respectively. All mice treated with mono- or dual-therapy succumbed to tumor burden. In contrast, mice treated with concurrent RT, PD-1 mAb and BGB-5777 (n = 9) showed a significant increase in median OS to 53 days (P Citation Format: Erik Ladomersky, Lijie Zhai, Galina Gritsina, Kristen L. Lauing, Matthew Genet, C. David James, Derek A. Wainwright. A novel IDO1 inhibitor combined with targeted immunotherapy durably increases survival in a mouse model of glioblastoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B018.