Meromit Singer

Purine synthesis promotes maintenance of brain tumor initiating cells in glioma

Brain tumor initiating cells (BTICs), also known as cancer stem cells, hijack high-affinity glucose uptake active normally in neurons to maintain energy demands. Here we link metabolic dysregulation in human BTICs to a nexus between MYC and de novo purine synthesis, mediating glucose-sustained anabolic metabolism. Inhibiting purine synthesis abrogated BTIC growth, self-renewal and in vivo tumor formation by depleting intracellular pools of purine nucleotides, supporting purine synthesis as a potential therapeutic point of fragility. In contrast, differentiated glioma cells were unaffected by the targeting of purine biosynthetic enzymes, suggesting selective dependence of BTICs. MYC coordinated the control of purine synthetic enzymes, supporting its role in metabolic reprogramming. Elevated expression of purine synthetic enzymes correlated with poor prognosis in glioblastoma patients. Collectively, our results suggest that stem-like glioma cells reprogram their metabolism to self-renew and fuel the tumor hierarchy, revealing potential BTIC cancer dependencies amenable to targeted therapy.

Induction and transcriptional regulation of the co-inhibitory gene module in T cells

The expression of co-inhibitory receptors, such as CTLA-4 and PD-1, on effector T cells is a key mechanism for ensuring immune homeostasis. Dysregulated expression of co-inhibitory receptors on CD4+ T cells promotes autoimmunity, whereas sustained overexpression on CD8+ T cells promotes T cell dysfunction or exhaustion, leading to impaired ability to clear chronic viral infections and diseases such as cancer1,2. Here, using RNA and protein expression profiling at single-cell resolution in mouse cells, we identify a module of co-inhibitory receptors that includes not only several known co-inhibitory receptors (PD-1, TIM-3, LAG-3 and TIGIT) but also many new surface receptors. We functionally validated two new co-inhibitory receptors, activated protein C receptor (PROCR) and podoplanin (PDPN). The module of co-inhibitory receptors is co-expressed in both CD4+ and CD8+ T cells and is part of a larger co-inhibitory gene program that is shared by non-responsive T cells in several physiological contexts and is driven by the immunoregulatory cytokine IL-27. Computational analysis identified the transcription factors PRDM1 and c-MAF as cooperative regulators of the co-inhibitory module, and this was validated experimentally. This molecular circuit underlies the co-expression of co-inhibitory receptors in T cells and identifies regulators of T cell function with the potential to control autoimmunity and tumour immunity.A module of co-inhibitory T cell receptors, driven by the cytokine IL-27, is identified in mice that is regulated by the transcription factors PRDM1 and c-MAF.

Effect of IL-7 Therapy on Phospho-Ribosomal Protein S6 and TRAF1 Expression in HIV-Specific CD8 T Cells in Patients Receiving Antiretroviral Therapy

IL-7 therapy has been evaluated in patients who do not regain normal CD4 T cell counts after virologically successful antiretroviral therapy. IL-7 increases total circulating CD4 and CD8 T cell counts; however, its effect on HIV-specific CD8 T cells has not been fully examined. TRAF1, a prosurvival signaling adaptor required for 4-1BB–mediated costimulation, is lost from chronically stimulated virus-specific CD8 T cells with progression of HIV infection in humans and during chronic lymphocytic choriomeningitis infection in mice. Previous results showed that IL-7 can restore TRAF1 expression in virus-specific CD8 T cells in mice, rendering them sensitive to anti–4-1BB agonist therapy. In this article, we show that IL-7 therapy in humans increases the number of circulating HIV-specific CD8 T cells. For a subset of patients, we also observed an increased frequency of TRAF1+ HIV-specific CD8 T cells 10 wk after completion of IL-7 treatment. IL-7 treatment increased levels of phospho-ribosomal protein S6 in HIV-specific CD8 T cells, suggesting increased activation of the metabolic checkpoint kinase mTORC1. Thus, IL-7 therapy in antiretroviral therapy–treated patients induces sustained changes in the number and phenotype of HIV-specific T cells.

Abstract A10: A distinct gene module for T cell dysfunction uncoupled from T cell activation and controlled by metallothioneins

Reversing the dysfunctional (also known as “exhausted”) T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients and for only some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we leveraged mouse models to analyze RNA profiles of CD8+ tumor-infiltrating lymphocytes (TILs) at various functional states. In a comparative study we identified metallothioneins, regulators of zinc metabolism, as drivers of T cell dysfunction and used genetic perturbation to identify, for the first time, a distinct gene module for T cell dysfunction that is uncoupled from T cell activation and associated with a CD8+ Treg signature. An analysis of TILs at single-cell resolution revealed that our dysfunction module negatively correlates with activation signatures also at the single-cell level, indicating that our uncoupled signatures for T cell dysfunction and activation can represent cell-intrinsic states. Clustering of the single-cells revealed a previously uncharacterized subpopulation of CD8+ TILs that was strongly enriched for our novel dysfunction signature, but not for activation signatures. This subpopulation was depleted of cells from a metallothionein KO mouse model in which T cell dysfunction was not observed, implying that we have indeed identified a phenotypically dysfunctional subpopulation. Using CRISPR/Cas9 genome editing we validate that Gata-3, a transcription factor expressed specifically in our identified subpopulation, is a driver of the dysfunctional phenotype in CD8+ TILs. Our results open novel avenues for specifically targeting the dysfunctional T cell state while leaving T cell activation programs intact. Citation Format: Meromit Singer, Chao Wang, Le Cong, Nemanja D. Marjanovic, Monika S. Kowalczyk, Huiyuan Zhang, Jackson Nyman, Kaori Sakuishi, Sema Kurtulus, David Gennert, Junrong Xia, John YH Kwon, James Nevin, Rebecca H. Herbst, Itai Yanai, Orit Rozenblatt-Rosen, Vijay K. Kuchroo, Aviv Regev, Ana C. Anderson. A distinct gene module for T cell dysfunction uncoupled from T cell activation and controlled by metallothioneins. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A10.

Abstract B085: Combining transcriptomic profiling and genome engineering to dissect regulation of tumor immunology

There is great interest in understanding the dysfunctional state of T-cells during uncontrolled tumor growth, particular regarding the molecular pathways that regulate this functional state for development of potential therapeutic approaches. Here we apply systematic transcriptomic profiling and analysis to dissect the molecular regulation of dysfunctional CD8+ tumor-filtrating lymphocytes (TILs) in a mice melanoma model. We identified key putative regulators and, through combination with precise genome perturbation, established their causal role in driving the dysfunctional phenotype of CD8+ TILs. Citation Format: Le Cong, Meromit Singer, Chao Wang, John Kwon, Ana Anderson, Orit Rozenblatt-Rosen, Vijay Kuchroo, Aviv Regev. Combining transcriptomic profiling and genome engineering to dissect regulation of tumor immunology [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 B085.