Systemic levels of methylmalonic acid (MMA), a byproduct of propionate metabolism, increase with age and MMA promotes tumor progression via its direct effects in tumor cells. However, the role of MMA in modulating the tumor ecosystem remains to be investigated. The proliferation and function of CD8+ T cells, key anti-tumor immune cells, declines with age and in conditions of vitamin B12 deficiency, which are the two most well-established conditions that lead to increased systemic levels of MMA. Thus, we hypothesized that increased circulatory levels of MMA would lead to a suppression of CD8+ T cell immunity. Treatment of primary CD8+ T cells with MMA induced a dysfunctional phenotype characterized by robust immunosuppressive transcriptional reprogramming and marked increases in the expression of the exhaustion regulator, TOX. Accordingly, MMA treatment upregulated exhaustion markers in CD8+ T cells and decreased their effector functions, which drove the suppression of anti-tumor immunity in vitro and in vivo. Mechanistically, MMA-induced CD8+ T cell exhaustion was associated with a suppression of NADH-regenerating reactions in the TCA cycle and concomitant defects in mitochondrial function. Thus, MMA has immunomodulatory roles, thereby highlighting MMA as an important link between aging, immune dysfunction, and cancer.

Abstract Purpose: Develop a shiny application to help integrate cancer datasets and guide researchers in selecting an appropriate method of correction for their technical artifacts. Description: Integrative analysis of heterogeneous expression data remains challenging due to variations in platform, RNA quality, sample processing, and other unknown technical effects. As the field performs omics profiling of samples from cancer patients and murine models, there is the need for harmonizing, identifying, and correcting these technical effects to ensure robust analysis on the treatment and conditional effects of the underlying genetics or biological events. However, selecting and implementing different approaches for removing unwanted batch effects can be a time-consuming and tedious process, especially for more biologically focused investigators. In this project, we present Shiny BATCH-FLEX, a Shiny app to rapidly visualize batch correction by established batch correction methods such as ComBat, Mean Centering, ComBatSeq, and Limma RemoveBatchEffect. With BATCH-FLEX, users can visualize the contribution of variance of a factor before and after correction using principal component analysis, relative log expression plots, heatmaps, and explanatory variables. Users can also save all plots and matrices as a single ZIP file for further downstream analysis. Results: As a proof of concept, we assessed BATCH-FLEX using simulated data generated from a linear model framework introduced by Gagnon-Bartsch and Speed, which assumes that gene expression measurements can be distilled to a combination of the biological signal, systemic nose, and random noise. BATCH-FLEX was able to successfully identify and remove the introduced effect using each of the batch correction methods listed above. Next, we evaluated BATCH-FLEX using a comprehensive collection of bladder cancer data consisting of microarray data from 13 studies spanning 1452 samples. Following the cleaning of study-dependent noise, BATCH-FLEX was successful in revealing the heterogeneity among bladder cancer based on known sample type annotations. Conclusion: We have developed BATCH-FLEX, a tool for oncologic researchers to rapidly assess, select, and implement commonly used batch correction methods. This tool is available at https://github.com/shawlab-moffitt/BATCH-FLEX. Our integrative web portal of a Bladder Cancer Resource for Translational Science (BEACON) will also be shared at the meeting. Citation Format: Joshua Davis, Alyssa Obermayer, Thac Duong, Rebecca Hesterberg, Xuefeng Wang, Mingxiang Teng, G. Daniel Grass, Timothy Shaw. BATCH-FLEX: Feature-level equalization of x-batch in heterogeneous cancer data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7423.

MOTIVATION:Integrative analysis of heterogeneous expression data remains challenging due to variations in platform, RNA quality, sample processing, and other unknown technical effects. Selecting the approach for removing unwanted batch effects can be a time-consuming and tedious process, especially for more biologically focused investigators. RESULTS:Here, we present BatchFLEX, a Shiny app that can facilitate visualization and correction of batch effects using several established methods. BatchFLEX can visualize the variance contribution of a factor before and after correction. As an example, we have analyzed ImmGen microarray data and enhanced its expression signals that distinguishes each immune cell type. Moreover, our analysis revealed the impact of the batch correction in altering the gene expression rank and single-sample GSEA pathway scores in immune cell types, highlighting the importance of real-time assessment of the batch correction for optimal downstream analysis. AVAILABILITY AND IMPLEMENTATION:Our tool is available through Github https://github.com/shawlab-moffitt/BATCH-FLEX-ShinyApp with an online example on Shiny.io https://shawlab-moffitt.shinyapps.io/batch_flex/.

Glutaminolysis is a hallmark of the activation and metabolic reprogramming of T cells. Isotopic tracer analyses of antigen-activated effector CD8+ T cells revealed that glutamine is the principal carbon source for the biosynthesis of polyamines putrescine, spermidine, and spermine. These metabolites play critical roles in activation-induced T cell proliferation, as well as for the production of hypusine, which is derived from spermidine and is covalently linked to the translation elongation factor eukaryotic translation initiation factor 5A (eIF5A). Here, we demonstrated that the glutamine/polyamine/hypusine axis controlled the expression of CD69, an important regulator of tissue-resident memory T cells (Trm). Inhibition of this circuit augmented the development of Trm cells ex vivo and in vivo in the BM, a well-established niche for Trm cells. Furthermore, blocking the polyamine/hypusine axis augmented CD69 expression as well as IFN-γ and TNF-α production in (a) human CD8+ T cells from peripheral blood and sarcoma tumor infiltrating lymphocytes and (b) human CD8+ CAR-T cells. Collectively, these findings support the notion that the polyamine-hypusine circuit can be exploited to modulate Trm cells for therapeutic benefit.

Lymphoma is a disease that principally afflicts older individuals and also impairs the immune system, yet how aging and cancer interact to affect the immune system remains poorly understood. This is particularly the case for malignancies that arise within hematopoietic tissues that closely interact with immune cells. We hypothesized that lymphoma-provoked changes to the adaptive immune system would differ based on age. To explore how the immune system is altered by both B-cell lymphoma and the aging process, we transplanted Eμ-Myc lymphoma B-cells into mice aged 6-12 weeks or 20-24 months. Interestingly, as marked by reduced splenomegaly and lymphoma cell numbers, tumor burden was significantly reduced in aged hosts, suggesting lymphoma cells grow more slowly in the aged tissue microenvironment. Further, while lymphoma progression provoked losses in total numbers of normal T-cells, NK-cells, and B-cells in young individuals, aged lymphocytes were surprisingly resistant to lymphoma. Similarly, profound phenotypic changes in T-cell subpopulations that are provoked by Eμ-Myc lymphoma in young individuals, such as increases in expression of CD44, CD69, PD-1, Foxp3, and Tox, were not manifest in lymphoma-bearing aged mice. In addition, immature NK cell population dynamics were altered by lymphomas differently among aged mice when compared to young mice. Finally, analyses of the chromatin state of enriched CD4+ T cells from young and aged mice by ATAC-seq revealed that the aging process provoked marked alterations in the epigenome, and that lymphoma only provoked significant changes to the epigenome among young CD4+ T cells. We conclude that aging-driven epigenetic alterations manifest in the adaptive immune system renders these cells surprisingly resilient to lymphoma-mediated immune suppression. Citation Format: Rebecca S. Hesterberg, Chia-Ho Cheng, Jiqiang Yao, Xiaofei Song, Xiaoqing Yu, John L. Cleveland. Aging dampens lymphoma-provoked phenotypic and epigenomic changes to the adaptive immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 621.

Background: Autologous chimeric antigen receptor (CAR) T-cell therapy is standard of care for patients with relapsed or refractory Large B cell Lymphoma (R/R LBCL). Each patient's CAR T product is unique, and the patient-specific determinants of CAR T-cell quality are poorly understood. CD39 is an ectonucleosidase that participates in the conversion of ATP and ADP and is upregulated in cancer. On T-cells, CD39 is expressed on CD4+ Tregs and on exhausted CD8+ T-cells that are prone to apoptosis. Methods: This is a retrospective single-center cohort study (N=72) of patients who received CD19 CAR T-cell therapy for LBCL (axi-cel n=52; tisa-cel n=20). Leukapheresis material was obtained from n=51 patients and flow cytometry was performed to phenotype CD4+ and CD8+ stem-central memory, memory, and effector T-cell subsets (using CCR7 and CD45RO), and for immune checkpoint expression (PD-1, LAG3, TIGIT, and CD39). 10x multiome (ATAC and RNA) single cell sequencing (scRNA-seq) was performed on n=8 leukapheresis products for deeper characterization. Serum metabolomics was performed on paired samples from n=21 patients. 10X Genomics Chromium scRNAseq was also performed on n=57 CAR T-cell infusion products, including n=36 with paired leukapheresis material previously phenotyped by flow cytometry. In vitro CAR T-cells were manufactured from starting patient T-cells and analyzed for CAR T-cell phenotype and function. Results: In the leukapheresis material of patients that did not achieve long-term remission after CAR T-cell therapy, and in those with high tumor burden, we found higher numbers of CD4+CD39+ and CD8+CD39+ T-cells ( Figs. 1 and 2). 10X multiome sequencing confirmed that CD39+ T-cells exhibit characteristics of exhaustion. Recent platinum-based chemotherapy did not affect CD39+ T-cell levels. In n=21 patients with paired serum analyzed by metabolomics, high CD8+CD39+ T-cells associated with reduced metabolites in the inosine-hypoxanthine pathway, which are downstream of the ectonucleotidase function of CD39 (inosine; P=0.006; hypoxanthine P=0.07). Increased numbers of CD39+ T-cells in the starting leukapheresis material translated into manufactured CAR T-cells having less favorable product characteristics, including higher CD39 expression and fewer memory cells in both paired patient samples and in experiments of in vitro CAR T-cell manufacturing. ScRNA-seq of the infused CAR T-cell product revealed differential gene signatures, both globally and in association with response, between axi-cel and tisa-cel CAR T-cell products, highlighting the profound product-specific differences. Nonetheless, both products were adversely affected by high CD39+ T-cells in the leukapheresis material used for manufacture. Conclusions: CD39+ T-cells, found in patients with high lymphoma tumor burden and an unfavourable immunometabolic environment, associate with poor CAR T-cell quality and adverse patient outcomes in R/R LBCL. Strategies are needed to improve patient T-cell quality prior to leukapheresis and/or improve CAR T-cell manufacturing from patients with high levels of exhausted CD39+ T-cells. Figure 1: Progression-free survival (PFS) stratified by leukapheresis CD8+CD39+ T-cells in the leukapheresis product. Blue - below cohort median percentage of CD8+T-cells that are CD39+ (low); Red - above median percentage of CD8+T-cells that are CD39+ (high). P-value by Log-Rank test. Overall survival (not shown) P-value 0.01. Figure 2: Proportion of CD8+CD39+ T-cells found in patients with low or high baseline metabolic tumor volume (MTV) based on a previously established cutoff (Dean et al. Blood Adv. 2020). MTV was measured on the pre-CAR T-cell PET/CT. P-value by T-test. MDJ, XY, and RMA are co-first authors with equal contribution.

CD8+ T cells play central roles in tumor immune surveillance and are major effectors in adoptive cell therapy (ACT). Thus, strategies that enhance their functions are an urgent clinical need. Metabolic reprogramming determines T cell fate and function, where glycolysis principally drives an effector phenotype while oxidative phosphorylation (OxPhos) drives T cell memory [1]. However, the roles of amino acid catabolism in controlling effector and memory CD8+ T cell fate and function are less well understood [1, 2]. Glutamine is essential for nucleotide biosynthesis and as an anaplerotic fuel source for the tricarboxylic acid (TCA) cycle. Isotope tracing experiments with 13C glutamine or 13C arginine revealed that glutamine is the major source of polyamines in activated CD8+ T cells. Further, activation of CD8+ T cells provokes marked increases in the expression and activity of enzymes that direct polyamine biosynthesis, specifically ornithine decarboxylase (Odc), spermidine synthase (Srm) and spermine synthase (Sms), as well as to increases in the polyamines putrescine, spermidine and spermine. Notably, pharmacologic inhibition of Odc using difluoromethylornithine (DFMO), or genetic deletion of Odc using CD8+ T cells from CD4-Cre;Odcfl/fl mice, augments cytokine production in activated CD8+ T cells, including IFN-γ, TNF-α and granzyme B. Further, metabolic flux analysis indicates that DFMO treatment switches the metabolism of activated CD8+ T cells to rely on OxPhos, a phenotype found in CD8+ tissue resident memory (TRM) cells that play key roles in tissue and anti-tumor immunity anti-tumor reactivity. Indeed, inhibition or loss of Odc augments other TRM phenotypes, including increases in CD69high;CD44high;S1PR1low;CD62Llow cells that define the TRM phenotype. In addition, ACT of DFMO treated CD8+ T cells augments their survival in vivo and augments the generation of CD69+CD103+CD69+CXCR6+ and Ly6C+ TRMs in the bone marrow. Finally, DFMO treatment enhances IFN-γ and TNF-α production in human TILs, and co-treatment with TGF-β polarizes TILs into CD69+CD103+ and CD69+CD49a+ TRM-like cells. We conclude that polyamines are a metabolic check point in CD8+ TRM generation and are an actionable target to improve the anti-tumor immunity of T-cell based immunotherapies. Citation Format: Aya G. Elmarsafawi, Rebecca S. Hesterberg, John L. Cleveland. Regulation and roles of polyamines in CD8+ T cell fate and function [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 1354.

Supplementary Table from TCR-Independent Metabolic Reprogramming Precedes Lymphoma-Driven Changes in T-cell Fate

Background CD8+ T cells play central roles in tumor immune surveillance and are major effectors in adoptive cell therapy. Thus, strategies that enhance their functions are an urgent clinical need. Metabolic reprogramming determines T cell fate and function.1 The roles of amino acid catabolism in controlling CD8+ T cell fate and function are less well understood.2,3 Glutamine is essential for nucleotide biosynthesis and as an anaplerotic fuel source for the tricarboxylic acid (TCA) cycle. Methods Isotope tracing experiments with 13C glutamine or 13C arginine Pharmacologic inhibition of ODC and DHPS Results Pharmacologic inhibition of Odc using difluoromethylornithine (DFMO), or inhibition of hypusination using the pharmacologic inhibitor of Dhps, GC7, augments cytokine production in activated CD8+ T cells, including IFN-γ and TNF-α. Indeed, inhibition of Odc or Dhps augments other TRM phenotypes, including increases in CD69high;S1PR1low;CD62Llow cells that define the tissue resident memory (TRM) CD8+ T cells that play key roles in tissue and anti-tumor immunity anti-tumor reactivity. In addition, ACT of DFMO treated CD8+ T cells augments their survival in vivo and augments the generation of CD69+CD103+CD69+CXCR6+ and Ly6C+ TRMs in the bone marrow. Finally, DFMO or GC7 treatment enhances IFN-γ and TNF-α production in human CD8 T cells. Conclusions We conclude that polyamine-hypusine axis is a metabolic check point in CD8+ TRM generation and are an actionable target to improve the anti-tumor immunity of T-cell based immunotherapies. Acknowledgements Metabolomics and Flow cytometry core at Moffitt Cancer Center References Sugiura A, and JC Rathmell. Metabolic Barriers to T Cell Function in Tumors. J Immunol, 2018;200(2): 400–407. Geiger R, et al. L-Arginine Modulates T cell metabolism and enhances survival and anti-tumor activity. Cell 2016. 167(3): 829–842.e13. Hesterberg RS, JL Cleveland, and PK Epling-Burnette. Role of polyamines in immune cell functions. Med Sci (Basel), 2018;6(1).

Ultraviolet radiation exposure (UVR) is a risk factor for cutaneous squamous cell carcinoma (cuSCC) and has been shown to be positively associated with circulating immunosuppressive regulatory T cells ("Tregs"). However, the risk of cuSCC in association with circulating Tregs has not been studied. The aim of this study was to determine whether circulating Treg levels are associated with cuSCC development, particularly in the context of high UVR. Blood and spectrophotometer-based UVR measurements were obtained on 327 immunocompetent individuals undergoing routine skin cancer screenings at baseline and followed for up to 4 years for incident cuSCC development within a prospective cohort study. Proportions of phenotypically distinct Tregs, especially CCR4hi and CLA+ cells which are associated with activation and homing, respectively, were measured by flow cytometry. Tregs in cuSCC tumors were assessed using immunohistochemistry and graded for solar elastosis, a measure of cumulative UVR damage. Of several Treg phenotypes examined, higher levels of circulating CCR4hi Tregs at baseline were significantly associated with increased risk of subsequent cuSCC; those with higher levels of both CCR4hi and UVR were four times more likely to develop cuSCC compared to those with lower levels of both (Hazard Ratio = 4.11, 95% CI = 1.22-13.90). Within cuSCC tumors, CCR4hi Tregs were positively associated with solar elastosis. Results show that a higher proportion of CCR4hi peripheral Tregs predicts incident cuSCC up to 4 years, especially among highly UV-exposed individuals. Research of the underpinning biology of Tregs in UVR-associated skin damage may possibly reveal novel opportunities for screening, prevention, and treatment.

Background: Ultraviolet radiation exposure (UVR) and immunosuppression are established risk factors for cutaneous squamous cell carcinoma (cuSCC). UVR can lead to systemic immune suppression as well as antigen specific immune tolerance due to generation of regulatory T cells (“Tregs”) which are known to have immunosuppressive effects. However, UVR-related immune responses in association with cuSCC risk have not been well studied in immunocompetent individuals. Objective: To determine whether circulating Tregs are associated with increased risk of cuSCC among individuals with higher UVR, we examined baseline Tregs, UVR, and subsequent cuSCC incidence among a subset of participants enrolled in a prospective cohort study of individuals undergoing skin cancer screening. Methods: 350 patients ages 60+ years with no prevalent skin cancer at the time of enrollment were recruited from the University of South Florida Dermatology Clinic in July 2014- July 2015 and followed for incident cuSCC up to four years later. At the baseline visit, blood was obtained, and a spectrophotometer was used to measure skin pigmentation at a sun-unexposed site (the axilla) and two sun-exposed sites (the forehead and the upper forearm). Recent UVR was quantified as the difference in readings between the sun-exposed and sun-unexposed sites. Using flow cytometry assays, lymphocyte samples were examined for Tregs (CD4+ CD25+ CD127low FoxP3+), and proportions of Tregs with enhanced immunosuppressive function (CD45RA−/CD27− Tregs) and skin tissue affinity (CLA+ Tregs, CCR4high Tregs) were determined. Associations between baseline Tregs and incident cuSCC were described using Cox Proportional Hazards, adjusting for age and sex, with and without stratification by higher versus lower levels of recent UVR. Results: Higher cuSCC incidence was observed in patients with higher circulating levels of CD45RA−/CD27− Tregs at baseline, as well as CLA+ Tregs, although the associations were not significant. Those with higher circulating CCR4hi Tregs at baseline had a 90% increased risk of developing a subsequent, incident cuSCC compared to those with lower levels (Hazard Ratio (HR)=1.90, 95% Confidence Interval (CI)= 1.03-3.49). Furthermore, participants with higher CCR4hi Tregs and higher UVR were four times more likely to develop an incident cuSCC compared to participants with lower CCR4hi Tregs and lower UVR (HR=4.11, 95% CI= 1.22-13.90). Conclusions: CCR4hi Tregs were associated with increased cuSCC risk, particularly among individuals with higher recent UVR exposure. CCR4hi Tregs may migrate out of circulation and into the skin in response to UVR-associated skin damage and possible release of local antigens, possibly influencing carcinogenesis by suppressing the immune response to UVR-damaged cells. Further research is needed to investigate the underpinning biology so that new opportunities for screening and prevention may be identified. Citation Format: Dana E. Rollison, Neil A. Fenske, Basil Cherpelis, Jane L. Messina, Yayi Zhao, Rossybelle P. Amorrortu, Rebecca Hesterberg, Pearlie K. Epling-Burnette. Circulating immunosuppressive regulatory T cells and risk of incident cutaneous squamous cell carcinoma [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 3492.

Immunomodulatory drugs, such as thalidomide and related compounds, potentiate T-cell effector functions. Cereblon (CRBN), a substrate receptor of the DDB1-cullin-RING E3 ubiquitin ligase complex, is the only molecular target for this drug class, where drug-induced, ubiquitin-dependent degradation of known "neosubstrates," such as IKAROS, AIOLOS, and CK1α, accounts for their biological activity. Far less clear is whether these CRBN E3 ligase-modulating compounds disrupt the endogenous functions of CRBN. We report that CRBN functions in a feedback loop that harnesses antigen-specific CD8+ T-cell effector responses. Specifically, Crbn deficiency in murine CD8+ T cells augments their central metabolism manifested as elevated bioenergetics, with supraphysiological levels of polyamines, secondary to enhanced glucose and amino acid transport, and with increased expression of metabolic enzymes, including the polyamine biosynthetic enzyme ornithine decarboxylase. Treatment with CRBN-modulating compounds similarly augments central metabolism of human CD8+ T cells. Notably, the metabolic control of CD8+ T cells by modulating compounds or Crbn deficiency is linked to increased and sustained expression of the master metabolic regulator MYC. Finally, Crbn-deficient T cells have augmented antigen-specific cytolytic activity vs melanoma tumor cells, ex vivo and in vivo, and drive accelerated and highly aggressive graft-versus-host disease. Therefore, CRBN functions to harness the activation of CD8+ T cells, and this phenotype can be exploited by treatment with drugs.

Cutaneous squamous cell carcinoma (cuSCC) accounts for 15-20% of skin cancers. Treatment of cuSCC with pramlintide, a synthetic analog of the hormone amylin, is currently under investigation in mouse models and human clinical trials. In cancer cells, pramlintide inhibits glycolysis, resulting in cell death and tumor regression. In order to investigate the potential use of pramlintide in combinatory therapies with checkpoint inhibitors, we aimed to study the immune modulatory effect of pramlintide in an immune competent mouse model of spontaneous ultraviolet radiation-induced cuSCC, as well as its effect on human T cells in vitro. To induce cuSCC, SKH-1 hairless mice were subjected to ultraviolet radiation until at least one tumor reached >4mm of diameter. Mice were then treated with 45 µg/kg of pramlintide or vehicle control for 10 days, every other day. Skin-draining lymph nodes, spleen and tumors were harvested for immunophenotyping of the T cell and myeloid compartment by flow cytometry. While the proportion of CD103+ dendritic cells (DCs) and CD3+ T cells remained constant after pramlintide treatment, a significant decrease in the proportion of both monocytic and granulocytic myeloid-derived suppressor cells (m-MDSCs and g-MDSCs) was observed relative to vehicle-control treated mice. Interestingly, pramlintide abolished m-MDSC differentiation from bone marrow progenitors in vitro suggesting that pramlintide may regulate a pathway necessary for m-MDSC development. Purified human CD3+ T cells activated in the presence of pramlintide exhibited a time-dependent increase in intracellular and secreted IFN-γ. Therefore, pramlintide may have immune modulatory effects based on both a reduction in myeloid-dependent suppression and a direct stimulatory effect on T cells that would culminate in superior anti-tumor T cell responses that augment immunotherapeutic approaches for the treatment of cuSCC. Citation Format: Leticia Tordesillas, Rebecca Hesterberg, Ivannie Ortiz-Rivera, Brittney R. Sell, Omar Chavez Chiang, Kimberly T. Nguyen, Brian L. Murphy, Pearlie K. Burnette, Elsa R. Flores, Kenneth Y. Tsai. Immune modulatory effect of pramlintide for cutaneous squamous cell carcinoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1227.

Approximately three million non-melanoma skin cancers (NMSCs) are diagnosed worldwide each year, although this number is likely an underestimate given that these cancers are not always recorded in cancer registries. Studies have suggested that skin (cutaneous) infections with human papillomaviruses (HPV) and polyomaviruses (HPyV) may play a role in the development of some NMSC types. Suppression of the immune system is also a risk factor for NMSC. This study, from the U.S.A, aimed to understand the relationship between T-regulatory (Treg) cells, cells which suppress immune response, and cutaneous viral infections. Blood, skin swabs and eyebrow hairs were collected from 352 patients who underwent skin cancer screening and did not have cancer detected. The researchers examined whether the skin swabs (SSW) and eyebrow hairs (EBH) contained genetic material (DNA) corresponding to 98 cutaneous HPV types (including beta HPV and gamma HPV) and 5 HPyV types. The blood samples were analyzed to determine proportions of different types of Treg cell populations in circulation (in the blood). The researchers found no association between total percent of circulating Treg cells and beta HPV or HPyV infection. However, two types of Treg cells were found at lower levels in those that had gamma HPV infection in their EBH and/or SSW. Those two types were CLA + Treg cells (known to travel to the skin) and effector CD27 - CD45RA - FOXP3 + CD4 + Treg cells (known to become active when exposed to a foreign viral infection). The study results suggest that gamma HPV infection may stimulate Treg cells to move from circulation into the skin tissues.

每年全世界约诊断出300万例非黑色素瘤皮肤癌(NMSC),尽管这个数字很可能被低估了,考虑到这类癌症不总是被记录在癌症注册数据库中。研究表明,人乳头瘤病毒 (HPV)和多瘤病毒(HPyV)皮肤感染可能对某些类型NMSC的出现起作用。免疫系统的抑制也是NMSC的一个风险因素。这项美国的研究旨在了解调节性T‐细胞(Treg)(抑制免疫反应的细胞)和病毒性皮肤感染之间的关系。从352名进行皮肤癌筛查且未检测到癌症的患者中收集血液、皮肤拭子和眉毛。研究者分析了皮肤拭子 (SSW) 和眉毛 (EBH) 是否含有 98 种皮肤 HPV(包括 β HPV 和 γ HPV)及 5 种 HPyV 相应的基因物质 (DNA)。还分析了血液样本,以确定在血液循环中不同类型T细胞群体的比例。研究发现,在血液循环 T 细胞的总比例和 β HPV 或 HPyV 感染之间无相关性。但发现在 EBH 和/或 SSW 出现 γ HPV 感染的患者中两种 T 细胞的水平更低。这两种 T细胞是 CLA+ T 细胞(已知可转运至皮肤)和CD27‐CD45RA‐FOXP3+CD4+效应T细胞(已知在暴露于外来病毒感染时激活)。研究结果表明,γHPV感染可能会刺激 T 细胞从血液循环转运至皮肤组织。

Abstract Therapeutic molecules targeting the activity of histone deacetylases (HDACs) are currently under investigation for the treatment of several malignancies. There are currently eighteen human HDACs and while histone deacetylation is associated with transcriptional repression, acetylated lysine targets are functionally diverse and include cytoplasmic, nuclear, and mitochondrial proteins. Here, we used a new class of novel small molecule inhibitors that are highly selective for HDAC11 to identify its role in the regulation of non-histone proteins. Stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry in the presence of HDAC11 selective inhibitors identified proteins with acetylation and/or expression changes after treatment and were compared to known HDAC substrates to establish a unique set of putative HDAC11 target proteins. Metabolic processes were highly enriched in this data set. Specifically, acetylated enolase 1 (ENO1, 2-phospho-D-glycerate hydrolase) which catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate (PEP) in the glycolytic pathway was highly altered after HDAC11 inhibition. Using acetylated lysine specific immunoprecipitation, we validated the hyperacetylated state of ENO1 upon HDAC11 inhibition. Functional assays confirmed that the HDAC11 inhibition lowered ENO1-mediated PEP production, and reduced proliferation and viability of hematopoietic and solid tumor cells. Similar observations were obtained in HDAC11 knock down cell lines confirming that HDAC11 is a required molecule in the regulation of ENO1-mediated metabolic regulation. The proteomics data also mapped three distinct target lysine residues of HDAC11 in ENO1 and each of these residues were substituted to either an acetylated or an un-acetylated lysine mimic to test their function in ENO1 activity and stability. We confirmed that K335 is the major target site of HDAC11 and its substitution to the acetylated mimic (glutamine) causes loss of enolase activity. Concomitantly, using proton nuclear magnetic resonance spectroscopy we identified some glycolytic intermediates upstream of ENO1 to be increased and downstream intermediates quantitatively reduced after HDAC11 inhibition suggesting that glycolysis is functionally suppressed. Glycolytic pathway disruption was associated with a compensatory increase in oxygen consumption and ATP production through oxidative phosphorylation in these oncogene transformed tumor cells, but not in their non-transformed counterparts. Suppression of fatty acid oxidation by inhibiting carnitine palmitoyltransferase 1 (CPT-1) or blocking glutamine utilization by inhibiting glutaminase (GLS1) in combination with HDAC11 inhibition resulted in a cooperative reduction in cellular ATP levels further supporting a direct role of HDAC11 in regulating glycolysis in tumor cells. For the first time, this study mechanistically and functionally defines a cytoplasmic non-histone protein regulated by HDAC11. Citation Format: Vasundhara Sharma, Agni Christodoulidou, Lanzhu Yue, Aileen Y. Alontaga, William E. Goodheart, Rebecca Hesterberg, Xiaozhang Zheng, Matthew W. Martin, Jennifer Y. Lee, Pearlie K. Burnette, Kenneth L. Wright. HDAC11 regulates lysine acetylation of enolase 1 [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 LB-249.

UV radiation (UVR) causing DNA damage is a well-documented risk factor for nonmelanoma skin cancer. Although poorly understood, UVR may also indirectly contribute to carcinogenesis by promoting immune evasion. To our knowledge, we report the first epidemiological study designed to investigate the association between quantitative measures of UVR, obtained using a spectrophotometer, and circulating T regulatory (Treg) cells. In addition to total Treg cells, the proportion of functionally distinct Treg cell subsets defined by CD45RA and CD27 phenotypic markers, graded expression of FOXP3 and CD25, and those expressing cutaneous lymphocyte–associated Ag and the chemokine receptor CCR4 were enumerated in 350 individuals undergoing routine skin cancer screening exams and determined not to have prevalent skin cancer. No associations were identified for UVR exposure or the overall proportion of circulating Treg cells; however, Treg cell subpopulations with an activation-associated phenotype, CD45RA−/CD27−, and those expressing cutaneous homing receptors were significantly positively associated with UVR. These subpopulations of Treg cells also differed by age, sex, and race. After stratification by natural skin tone, and adjusting for age and sex, we found that spectrophotometer-based measures of UVR exposure, but not self-reported measures of past sun exposure, were positively correlated with the highest levels of these Treg cell subpopulations, particularly among lighter-skinned individuals. Findings from this large epidemiologic study highlight the diversity of human Treg cell subpopulations associated with UVR, thus raising questions about the specific coordinated expression of CD45RA, CD27, CCR4, and cutaneous lymphocyte–associated Ag on Treg cells and the possibility that UVR contributes to nonmelanoma skin cancer carcinogenesis through Treg cell–mediated immune evasion.

Cutaneous viral infections and immune suppression are risk factors for some forms of nonmelanoma skin cancer; however, their interrelationship is poorly understood.

Immunomodulatory drugs, such as lenalidomide, pomalidomide, and CC122 are glutarimide derivatives that alter T cell effector functions, reverse tolerance, and increase cytokine production. We hypothesized that cereblon (CRBN), the molecular target of these drugs, is a negative regulator of T cell function. Therefore, germline Crbn knockout mice were used to investigate its role in T cell regulation. Similar to human T cells treated with immunomodulatory drugs, stimulated Crbn−/− T cells exhibit increased proliferation and cytokine production and maintain this phenotype in the absence of CD28 co-stimulation. Using Gene Set Enrichment Analysis (GSEA) to identify transcriptional drivers of differentially expressed genes, we found that Myc and its family member Max may drive the observed activated phenotype in Crbn−/− T cell. Crbn−/− T cells have Myc-related phenotypic changes, such as increased cell size, CD98 expression, glucose uptake, glutamine and arginine uptake, increased intracellular polyamine biosynthesis, and oxygen consumption. Similar increases in Myc-related processes were observed in activated human T cells treated with immunomodulatory drugs. While c-myc mRNA is similar, Crbn−/− T cells and drug-treated human T cells have prolonged c-Myc protein expression. CRBN, a substrate receptor for the DDB1/Cul4A/Rbx1 E3 ubiquitin ligase complex, has only one known endogenous substrate which is glutamine synthetase (GS). GS interacts with CRBN through acetylated lysine residues yielding a protein that is succeptable to polyubiquitinlyation. Collectively, this suggests that CRBN tunes c-Myc-regulated pathways in activated T cells and that this process is blocked by immunomodulatory drugs.