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Adoptive T cell therapies (ACT) have been curative for a limited number of cancer patients. The sensitization of cancer cells to T cell killing may expand the benefit of these therapies for more patients. To this end, we use a three-step approach to identify cancer genes that disfavor T cell immunity. First, we profile gene transcripts upregulated by cancer under selection pressure from T cell killing. Second, we identify potential tumor gene targets and pathways that disfavor T cell killing using signaling pathway activation libraries and genome-wide loss-of-function CRISPR-Cas9 screens. Finally, we implement pharmacological perturbation screens to validate these targets and identify BIRC2, ITGAV, DNPEP, BCL2, and ERRα as potential ACT-drug combination candidates. Here, we establish that BIRC2 limits antigen presentation and T cell recognition of tumor cells by suppressing IRF1 activity and provide evidence that BIRC2 inhibition in combination with ACT is an effective strategy to increase efficacy.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade.Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade.CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo.CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy.This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

The administration of T cells as cellular therapy against advanced cancers has brought clinical benefit to many patients and has progressed the field of cancer research. However, current cell therapy treatments are not curative in most patients, particularly in those with solid tumors, and it remains to be seen how broadly and efficaciously they may be applied going forward. Recent research has begun to elucidate key factors that regulate the efficacy of cell therapy in cancer patients, including T cell stemness and the ability to effectively target tumor antigens and overcome tumor heterogeneity. In this review, we discuss key properties of clinically effective anti-cancer T cell therapies along with strategies to improve T cell characteristics to augment clinical efficacy in solid tumors.

Lee et al. reveal that administration of IL-1β can alter the host environment to augment the magnitude and functionality of CD8+ T cells, thereby improving the efficacy of adoptively transferred tumor-reactive T cells in treating large, established tumors in mice., Host conditioning has emerged as an important component of effective adoptive cell transfer–based immunotherapy for cancer. High levels of IL-1β are induced by host conditioning, but its impact on the antitumor function of T cells remains unclear. We found that the administration of IL-1β increased the population size and functionality of adoptively transferred T cells within the tumor. Most importantly, IL-1β enhanced the ability of tumor-specific T cells to trigger the regression of large, established B16 melanoma tumors in mice. Mechanistically, we showed that the increase in T cell numbers was associated with superior tissue homing and survival abilities and was largely mediated by IL-1β–stimulated host cells. In addition, IL-1β enhanced T cell functionality indirectly via its actions on radio-resistant host cells in an IL-2– and IL-15–dependent manner. Our findings not only underscore the potential of provoking inflammation to enhance antitumor immunity but also uncover novel host regulations of T cell responses.

SUMMARYThe human definitive yolk sac is an important organ supporting the early developing embryo through nutrient supply and by facilitating the establishment of the embryonic circulatory system. However, the molecular and cellular biology of the human yolk sac remains largely obscure due to the lack of suitable in vitro models. Here, we show that human induced pluripotent stem cells (hiPSCs) co-cultured with various types of stromal cells as spheroids self-organize into yolk sac-like organoids without the addition of exogenous factors. Yolk sac-like organoids recapitulated a yolk sac specific cellular complement and structures as well as the functional ability to generate definitive hematopoietic progenitor cells (HPCs). Furthermore, sequential hemato-vascular ontogenesis could be observed during organoid formation. Notably, our organoid system can be performed in a scalable, autologous, and xeno-free condition, thereby providing an important model of human definitive yolk sac development and allows for efficient bulk generation of hiPSC-derived HPCs.

Background Neoantigen-specific T cells isolated from tumors have shown promise clinically but fail to consistently elicit durable tumor regression. Expression of the intracellular checkpoint CISH is elevated in human tumor infiltrating lymphocytes (TIL) and has been shown to inhibit neoantigen reactivity in murine TIL. Methods To explore CISH function in human T cells we developed a CRISPR/Cas9-based strategy to knockout (KO) CISH in human T cells with high-efficiency (>90%) and without detectable off-target editing. Results CISH KO in peripheral blood T cells enhanced proliferation, cytokine polyfunctionality, and cytotoxicity in vitro. To determine if CISH KO similarly enhances TIL function, we developed a clinical-scale, GMP-compliant manufacturing process for CISH disruption in primary human TIL. In process validation runs we achieved CISH KO efficiencies >90% without detectable off-target editing while maintaining high viability and expansion. Compared to WT controls, CISH KO in patient-derived TIL demonstrated increased proliferation, T cell receptor (TCR) avidity, neoantigen recognition, and unmasked reactivity to common p53 mutations. Hyperactivation in CISH KO TIL did not increase differentiation, suggesting that CISH KO may uncouple activation and differentiation pathways. Single cell profiling identifies a pattern of CISH expression inverse to key regulators of activation, and CISH KO in human TIL increases PD1 expression. Adoptive transfer of Cish KO T cells synergistically combines with PD1 inhibition resulting in durable tumor regression in mice, highlighting orthogonal dual cell surface and intracellular checkpoint inhibition as a novel combinatorial approach for T cell immunotherapy. Conclusions These pre-clinical data offer new insight into neoantigen recognition and serve as the basis for a recently initiated human clinical trial at the University of Minnesota (NCT04426669) evaluating inhibition of the novel intracellular immune checkpoint CISH in a CRISPR-engineered, neoantigen-specific T cell therapy for solid tumors. Updates from the clinical trial will be highlighted. Trial Registration NCT04426669

While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLCγ-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

Although immunotherapy has revolutionized cancer treatment, only a subset of patients demonstrate durable clinical benefit. Definitive predictive biomarkers and targets to overcome resistance remain unidentified, underscoring the urgency to develop reliable immunocompetent models for mechanistic assessment. Here we characterize a panel of syngeneic mouse models, rep-resenting a variety of molecular and phenotypic subtypes of human melanomas and exhibiting their diverse range of responses to immune checkpoint blockade (ICB). Comparative analysis of genomic, transcriptomic and tumor-infiltrating immune cell profiles demonstrated alignment with clinical observations and validated the correlation of T cell dysfunction and exclusion programs with resistance. Notably, genome-wide expression analysis uncovered a melanocytic plasticity signature predictive of patient outcome in response to ICB, suggesting that the multipotency and differentiation status of melanoma can determine ICB benefit. Our comparative preclinical platform recapitulates melanoma clinical behavior and can be employed to identify mechanisms and treatment strategies to improve patient care.

Small molecule based targeted therapies for the treatment of metastatic melanoma hold promise but responses are often not durable, and tumors frequently relapse. Response to adoptive cell transfer (ACT)-based immunotherapy in melanoma patients are durable but patients develop resistance primarily due to loss of antigen expression. The combination of small molecules that sustain T cell effector function with ACT could lead to long lasting responses. Here, we have developed a novel co-culture cell-based high throughput assay system to identify compounds that could potentially synergize or enhance ACT-based immunotherapy of melanoma. A BRAFV600E mutant melanoma cell line, SB-3123p which is resistant to Pmel-1-directed ACT due to low gp100 expression levels was used to develop a homogenous time resolve fluorescence (HTRF), screening assay. This high throughput screening assay quantitates IFNγ released upon recognition of the SB-3123p melanoma cells by Pmel-1 CD8+ T-cells. A focused collection of approximately 500 small molecules targeting a broad range of cellular mechanisms was screened, and four active compounds that increased melanoma antigen expression leading to enhanced IFNγ production were identified and their in vitro activity was validated. These four compounds may provide a basis for enhanced immune recognition and design of novel therapeutic approaches for patients with BRAF mutant melanoma resistant to ACT due to antigen downregulation.

While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLCγ-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

Cancer recurrence after initial diagnosis and treatment is a major cause of breast cancer (BC) mortality, which results from the metastatic outbreak of dormant tumour cells. Alterations in the tumour microenvironment can trigger signalling pathways in dormant cells leading to their proliferation. However, processes involved in the initial and the long-term survival of disseminated dormant BC cells remain largely unknown. Here we show that autophagy is a critical mechanism for the survival of disseminated dormant BC cells. Pharmacologic or genetic inhibition of autophagy in dormant BC cells results in significantly decreased cell survival and metastatic burden in mouse and human 3D in vitro and in vivo preclinical models of dormancy. In vivo experiments identify autophagy gene autophagy-related 7 (ATG7) to be essential for autophagy activation. Mechanistically, inhibition of the autophagic flux in dormant BC cells leads to the accumulation of damaged mitochondria and reactive oxygen species (ROS), resulting in cell apoptosis., Highly metastatic dormant cancer cells contribute to breast cancer recurrence, but the underlying mechanism is less understood. Here, the authors show that dormant breast cancer cells depend on autophagy to ensure their long term survival and distant recurrence

T cells play a central role in cancer immunosurveillance and current cancer immunotherapies, including adoptive cell transfer (ACT), T cell receptor or chimeric antigen receptor (CAR) T cell therapies and immune checkpoint blockade. Understanding the factors regulating T cell function is hence critical for improving the success of these immunotherapies. It has been recognized that metabolism can greatly affect different aspects of T cell function, including differentiation, cytokine production, longevity and exhaustion. Here we integrate genome-scale metabolic modeling (GEM) with biological experiments to discover novel metabolic determinants of T cell function. Previously we developed the metabolic transformation algorithm (MTA), a GEM method that was successfully applied to identify driving factors and targets for different diseases. Here applying MTA to data on CAR-T cell gene expression and patient response to anti-CD19 CAR-T therapy, we predicted mitochondrial metabolite transport and specifically proton transport in mitochondrial uncoupling, as key determinants of CAR-T therapy response. Mitochondrial uncoupling is also important for the in vivo persistence of adoptively transferred tumor-infiltrating lymphocytes, as further confirmed by analyzing their gene expressions from a KRAS-targeting ACT dataset. Focusing on the mitochondrial uncoupling protein 2 (Ucp2), which is abundantly expressed in T cells, we experimentally validated that it is required for T cell longevity and anti-tumor function. Specifically, the loss of Ucp2 either via knock-out (KO) or treatment by genipin (a Ucp2 inhibitor) in mice T cells results in accelerated differentiation into “terminal effector cells”, as shown by increased levels of T cell cytotoxicity and exhaustion markers, and decreased levels of central memory and stemness markers. Adoptive transfer of Ucp2-KO Pmel-1 T cells to mice bearing B16 melanomas displayed poorer anti-tumor efficacy and worse survival than the transfer of Ucp2-wildtype T cells. We find that Ucp2 modulates oxidative stress and DNA damage by regulating the levels of mitochondrial superoxide. Reducing mitochondrial reactive oxygen species was sufficient to rescue the loss of Ucp2-mediated effector T cell differentiation, senescence, cytokine production and anti-tumor activity Ucp2-KO Pmel-1 T cells. Tumor-specific CD8+ T cells could be metabolically reprogrammed by Ucp2 overexpression, which improved T cell longevity and anti-tumor function in the Pmel-1/B16 ACT mice model. In sum, our study establishes a novel role of Ucp2 in regulating T cell longevity and anti-tumor activity by repressing increased ROS levels accompanying mitochondrial dysfunction in differentiated and exhausted cells, suggesting that manipulating Ucp2 levels in T cells can be exploited to enhance T cell-based cancer immunotherapies. Citation Format: Madhusudhanan Sukumar, Kuoyuan Cheng, Arunakumar Gangaplara, Yogin Patel, Suman K. Vodnala, Rafiqul Islam, Arash Eidizadeh, Carolyn Subramaniam, Ping Lee, Rigel Kishton, Amanda N. Henning, Michael J. Kruhlak, Zhiya Yu, Ethan M. Shevach, Toren Finkel, Eytan Ruppin, Nicholas P. Restifo. Integrated computational and experimental analysis identifies the mitochondrial uncoupling protein 2 (Ucp2) as a key regulator of T cell anti-tumor function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1527.

Immune cells called cytotoxic T cells can recognize and destroy cancer cells. The finding that stem-cell-like T cells exist in tumours, at niche sites that support these cells, could aid efforts to boost anticancer immune responses. Stem-cell-like T cells reside in niches found in tumours.

SummaryAll current highly effective anti-tumor immunotherapeutics depend on the activity of T cells, but tumor cells can escape immune recognition by several mechanisms including loss of function in antigen presentation and inflammatory response genes, expression of immunomodulatory proteins and an immunosuppressive tumor microenvironment. In contrast, the comprehensive identification of strategies that sensitize tumor cells to immunotherapy in vivo has remained challenging. Here, we combine a two-cell type (2CT) whole-genome CRISPR-Cas9 screen with dynamic transcriptional analysis (DTA) of tumor upon T cell encounter to identify a set of genes that tumor cells express as an active defense against T cell-mediated killing. We then employed small molecule and biologic screens designed to antagonize gene products employed by tumor cells to actively defend against T cell-mediated tumor destruction and found that the inhibition of BIRC2, ITGAV or DNPEP enhanced tumor cell destruction by T cells. Mechanistically, we found that BIRC2 promoted immunotherapy resistance through inhibiting non-canonical NF-κB signaling and limiting inflammatory chemokine production. These findings show the path forward to improving T cell-mediated tumor destruction in the clinic.

Although immunotherapy has revolutionized cancer treatment, only a subset of patients demonstrates durable clinical benefit. Definitive predictive biomarkers and targets to overcome resistance remain unidentified, underscoring the urgency to develop reliable immunocompetent models for mechanistic assessment. Here we characterize a panel of syngeneic mouse models representing the main molecular and phenotypic subtypes of human melanomas and exhibiting their range of responses to immune checkpoint blockade (ICB). Comparative analysis of genomic, transcriptomic and tumor-infiltrating immune cell profiles demonstrated alignment with clinical observations and validated the correlation of T cell dysfunction and exclusion programs with resistance. Notably, genome-wide expression analysis uncovered a melanocytic plasticity signature predictive of patient outcome in response to ICB, suggesting that the multipotency and differentiation status of melanoma can determine ICB benefit. Our comparative preclinical platform recapitulates melanoma clinical behavior and can be employed to identify new mechanisms and treatment strategies to improve patient care.

Across clinical trials, T cell expansion and persistence following adoptive cell transfer (ACT) have correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis to identify actionable ligand-receptor pairs capable of compromising T cell durability following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments (TMEs). Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T cells used for clinical ACT. We hypothesized that a cognate Fas-FasL interaction within the TME might limit both T cell persistence and antitumor efficacy. We discovered that genetic engineering of Fas variants impaired in the ability to bind FADD functioned as dominant negative receptors (DNRs), preventing FasL-induced apoptosis in Fas-competent T cells. T cells coengineered with a Fas DNR and either a T cell receptor or chimeric antigen receptor exhibited enhanced persistence following ACT, resulting in superior antitumor efficacy against established solid and hematologic cancers. Despite increased longevity, Fas DNR-engineered T cells did not undergo aberrant expansion or mediate autoimmunity. Thus, T cell-intrinsic disruption of Fas signaling through genetic engineering represents a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies.

Breast cancer is the second leading cause of cancer-related deaths in the United States, with the majority of these deaths due to metastatic lesions rather than the primary tumor. Thus, a better understanding of the etiology of metastatic disease is crucial for improving survival. Using a haplotype mapping strategy in mouse and shRNA-mediated gene knockdown, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor. We found that the role of Rnaseh2c in metastatic disease is independent of RNase H2 enzymatic activity, and immunophenotyping and RNA-sequencing analysis revealed engagement of the T cell-mediated adaptive immune response. Furthermore, the cGAS-Sting pathway was not activated in the metastatic cancer cells used in this study, suggesting that the mechanism of immune response in breast cancer is different from the mechanism proposed for Aicardi-Goutières Syndrome, a rare interferonopathy caused by RNase H2 mutation. These results suggest an important novel, non-enzymatic role for RNASEH2C during breast cancer progression and add Rnaseh2c to a panel of genes we have identified that together could determine patients with high risk for metastasis. These results also highlight a potential new target for combination with immunotherapies and may contribute to a better understanding of the etiology of Aicardi-Goutières Syndrome autoimmunity., Author summary The majority of breast cancer-associated deaths are due to metastatic disease, the process where cancerous cells leave the primary tumor in the breast and spread to a new location in the body. To better understand the etiology of this process, we investigate the effects of gene expression changes in the primary tumor. In this study, we found that changing the expression of the gene Rnaseh2c changed the number of metastases that developed in the lungs of tumor-bearing mice. By investigating the enzyme complex Rnaseh2c is part of, RNase H2, we determined that Rnaseh2c’s effects may be independent of RNase H2 enzyme activity. Because Rnaseh2c is known to cause the autoimmune disease Aicardi-Goutières Syndrome (AGS), we tested whether the immune system is involved in the metastatic effect. Indeed, we found that the cytotoxic T cell response helps mediate the effect that Rnaseh2c has on metastasis. Together these data indicate that Rnaseh2c expression contributes to a patient’s susceptibility to developing breast cancer metastasis and demonstrate that the immune system is involved in this outcome. The implications of this study suggest immunotherapy could be a viable treatment for breast cancer metastasis and may help inform the understanding of AGS and RNase H2 in cancer.

Summary T cells are central to all currently effective cancer immunotherapies, but the characteristics defining therapeutically effective anti-tumor T cells have not been comprehensively elucidated. Here, we delineate four phenotypic qualities of effective anti-tumor T cells: cell expansion, differentiation, oxidative stress, and genomic stress. Using a CRISPR-Cas9-based genetic screen of primary T cells we measured the multi-phenotypic impact of disrupting 25 T cell receptor-driven kinases. We identified p38 kinase as a central regulator of all four phenotypes and uncovered transcriptional and antioxidant pathways regulated by p38 in T cells. Pharmacological inhibition of p38 improved the efficacy of mouse anti-tumor T cells and enhanced the functionalities of human tumor-reactive and gene-engineered T cells, paving the way for clinically relevant interventions.

Stemness against adversity T lymphocytes are powerful immune cells that can destroy tumors, but cancers have developed tricks to evade killing. Vodnala et al. found that potassium ions in the tumor microenvironment serve a dual role of influencing T cell effector function and stemness (see the Perspective by Baixauli Celda et al. ). Increased potassium impairs T cell metabolism and nutrient uptake, resulting in a starvation state known as autophagy. The increased potassium can also preserve T cells in a stem-like state where they retain the capacity to divide. These seemingly divergent processes are linked to the cellular distribution of acetyl–coenzyme A, which, when manipulated, can restore the ability of human T cells to eliminate tumors in mice. Science , this issue p. eaau0135 ; see also p. 1395

Adoptive immunotherapies using T cells genetically redirected with a chimeric antigen receptor (CAR) or T cell receptor (TCR) are entering mainstream clinical practice. Despite encouraging results, some patients do not respond to current therapies. In part, this phenomenon has been associated with infusion of reduced numbers of early memory T cells. Herein, we report that AKT signaling inhibition is compatible with CAR and TCR retroviral transduction of human T cells while promoting a CD62L-expressing central memory phenotype. Critically, this intervention did not compromise cell yield. Mechanistically, disruption of AKT signaling preserved MAPK activation and promoted the intranuclear localization of FOXO1, a transcriptional regulator of T cell memory. Consequently, AKT signaling inhibition synchronized the transcriptional profile for FOXO1-dependent target genes across multiple donors. Expression of an AKT-resistant FOXO1 mutant phenocopied the influence of AKT signaling inhibition, while addition of AKT signaling inhibition to T cells expressing mutant FOXO1 failed to further augment the frequency of CD62L-expressing cells. Finally, treatment of established B cell acute lymphoblastic leukemia was superior using anti-CD19 CAR-modified T cells transduced and expanded in the presence of an AKT inhibitor compared with conventionally grown T cells. Thus, inhibition of signaling along the PI3K/AKT axis represents a generalizable strategy to generate large numbers of receptor-modified T cells with an early memory phenotype and superior antitumor efficacy.

Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR-Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8+ T cells with the resistance or non-responsiveness of cancer to immunotherapies.

New drugs for the treatment of human African trypanosomiasis are urgently needed. A number of 2-aminopyrazines/2-aminopyridines were identified as promising leads following a focused screen of 5,500 compounds for Trypanosoma brucei subsp. brucei viability. Described compounds are trypanotoxic in the submicromolar range and show comparably low cytotoxicity on representative mammalian cell lines. Specifically, 6-([6-fluoro-3,4-dihydro-2 H -1-benzopyran-4-yl)]oxy)- N -(piperidin-4-yl)pyrazin-2-amine (CBK201352) is trypanotoxic for T. brucei subsp. brucei , T. brucei subsp. gambiense , and T. brucei subsp. rhodesiense and is nontoxic to mammalian cell lines, and in vitro preclinical assays predict promising pharmacokinetic parameters. Mice inoculated intraperitoneally (i.p.) with 25 mg/kg CBK201352 twice daily for 10 days, starting on the day of infection with T. brucei subsp. brucei , show complete clearance of parasites for more than 90 days. Thus, CBK201352 and related analogs are promising leads for the development of novel treatments for human African trypanosomiasis.

Tumours progress despite being infiltrated by tumour-specific effector T cells1. Tumours contain areas of cellular necrosis, which is associated with poor survival in a variety of cancers2. Here, we show that necrosis releases an intracellular ion, potassium, into the extracellular fluid of mouse and human tumours causing profound suppression of T cell effector function. We find that elevations in the extracellular potassium concentration [K+]e act to impair T cell receptor (TCR)-driven Akt-mTOR phosphorylation and effector programmes, this potassium-mediated suppression of Akt-mTOR signalling and T cell function is dependent upon the activity of the serine/threonine phosphatase PP2A3,4. While the suppressive effect mediated by elevated [K+]e is independent of changes in plasma membrane potential (Vm), it does require an increase in intracellular potassium ([K+]i). Concordantly, ionic reprogramming of tumour-specific T cells through overexpression of the potassium channel Kv1.3 lowers [K+]i and improves effector functions in vitro and in vivo. Consequently, Kv1.3 T cell overexpression enhances tumour clearance and survival of melanoma-bearing mice. These results uncover a previously undescribed ionic checkpoint blocking T cell function within tumours and identify new strategies for cancer immunotherapy.

Infections with subspecies of the extracellular parasite Trypanosoma brucei cause African trypanosomiasis, a disease that affects both humans (sleeping sickness) and animals. During the early stage of human African trypanosomiasis the parasites invade the hemolymphatic system, and during the late meningoencephalitic stage severe signs of nervous system involvement are observed [1–3]. In a mouse model of the disease, T. brucei brucei penetrate the blood-brain barrier (BBB) at a late stage and can enter the brain parenchyma [4]. Within the CNS, activation of white blood cell infiltrates and resident cells probably leads to the nervous system disease [5]. The innate immune system has evolved several strategies of self-nonself discrimination that are based on the recognition of molecular patterns demarcating infectious nonself. Different Toll-like receptors (TLRs), by recognizing diverse pathogen-associated molecular patterns of microbes, activate innate immunity and may initiate the subsequent development of adaptive immunity. TLR agonists stimulate the secretion of proinflammatory cytokines and type I interferons (IFN-α/β) that are involved not only in protection against infections but also in infection-mediated pathology. The development of adaptive immune responses is at least in part mediated through the ability of the innate receptor signaling to activate and stimulate the migration of antigen-presenting cells into the lymph nodes [6]. The binding of TLRs (except TLR3) by their corresponding ligands results in the recruitment of the intracellular adaptor molecule MyD88. Myd88–/– animals are highly susceptible to infection with a wide variety of different pathogens, including infection with T. brucei brucei [7–9]. We have shown elsewhere that the T-cell–derived cytokine IFN-γ, as well as the IFN-inducible chemokine CXCL10, promote the penetration of T cells and parasites in the brain [4, 10], suggesting also that parasites follow T cells during their brain invasion across the BBB. Despite the accepted view that signaling from specific innate immune receptors are required to activate and determine the quality of T-cell responses, the role of innate immunity in T-cell–mediated central nervous system (CNS) diseases, such as sleeping sickness, is poorly understood. In the current study, we investigated whether signals emanating from TLR control the accumulation of T cells and parasites in the brain parenchyma. We demonstrate that TLR signaling regulates the penetration of T cells and parasites across the BBB and controls survival of the latter in the brain parenchyma through distinct mechanisms.

Cancer recurrence resulting from the metastatic outbreak of dormant disseminated tumor cells following the apparent successful treatment of the primary tumor is a major cause of breast cancer mortality. However, little is known regarding the molecular mechanisms governing tumour cell dormancy and the dormant-to-proliferative switch, impeding the development of effective therapeutic strategies. We therefore investigated whether stress-induced autophagy may promote survival of dormant cancer cells and, consequently, inhibition of autophagy could prevent breast cancer recurrence. To address the functional role of autophagy in breast cancer progression and the potential therapeutic impact of its inhibition, we utilized mouse and human 3D in vitro and in vivo preclinical models of dormancy. The analysis of autophagy markers and use of an autophagic flux biosensor allowed direct visualization of autophagic vesicles and their evolution in breast cancer dormant cells over time. In agreement with our hypothesis, pharmacologic or genetic inhibition of autophagy in dormant breast cancer cells resulted in significantly decreased cell survival and metastatic burden in vitro and in vivo. Furthermore, the inhibition of autophagy prevented the spontaneous dormant-to-proliferative switch of highly metastatic cells. In contrast, proliferating disseminated cells were insensitive to autophagy blockade. Indeed, in vivo analysis of the autophagic flux over time confirmed that autophagy is a critical survival process activated and maintained in dormant breast cancer cells, which is shut down after the cells undergo the dormant-to-proliferative switch. Transcriptomic analysis and in vivo metastatic burden assays identified the autophagy-related 7 (ATG7) gene, but not Beclin1 (BECN1), to be essential for autophagy activation, indicating that a non-canonical autophagy pathway is activated in dormant breast cancer cells. Co-localization studies identified mitochondria as the predominant autophagosomal cargo in breast cancer dormant cells. Mechanistically, inhibition of the autophagic flux in dormant breast cancer cells led to the accumulation of depolarized mitochondria and reactive oxygen species (ROS), resulting in cell apoptosis. This study has important implications regarding the role of autophagy in breast cancer progression and suggests that inhibition of autophagy may be of therapeutic value in preventing breast cancer recurrence. Furthermore, it provides novel insights into the molecular mechanisms for survival of breast cancer dormant cells. Citation Format: Laura Vera Ramirez, Suman K. Vodnala, Ryan Nini, Kent W. Hunter, Jeffrey E. Green. Survival of dormant breast cancer cells and metastatic tumor recurrence is dependent upon the activation of autophagy [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 57.

Melanoma is an aggressive and lethal disease with no efficacious therapies for a broad subset of late stage patients. Current immunotherapies, including immune checkpoint blockade (ICB), may prolong survival in certain patients, but generate responses in less than 40% of the treated cohort. This demands a better understanding of molecular mechanisms underlying the lack of response and acquired resistance to ICB. However, functional studies are limited in patients, and current preclinical studies are handicapped by the absence of appropriate mouse models that recapitulate the pathological and immunological diversity of human melanomas. Here we develop four syngeneic melanoma mouse models with human-relevant genetic modifications and carcinogenic agents, which we hypothesize will mirror the spectrum of responses to ICB and offer a platform for future mechanistic studies in melanoma. The models are: 1) neonatal ultraviolet radiation (UV)-induced melanoma in a HGF-transgenic mouse, in which melanocyte localization at the epidermal-dermal junction mimics human distribution (HU); 2) 7,12-Dimethylbenz(a)anthracene (DMBA)-induced melanoma in a HGF-tg and Cdk4R24C mouse (HC4D); 3) UV-induced melanoma in a BrafCA/+; HGF-tg; Cdkn2aflox/+; Tyr-CreERT2-t mouse (BHCU); and 4) UV-induced melanoma in a BrafCA/+; Ptenflox/+; Cdkn2aflox/+; Tyr-CreERT2-tg mouse (BPCU). Exome sequencing of the four models reveals a high correlation with mutational subtypes previously described in human melanoma. BPCU and BHCU represent different Braf mutant patient populations and HU and HC4D represent triple wildtype melanoma (non-BRAF, -NRAS, -NF1). The four mouse models demonstrate distinct responses to ICB with anti-CTLA-4 treatment. While HU and HC4D melanomas show high or partial sensitivity to anti-CTLA-4, respectively, BPCU and BHCU do not respond to treatment. In vivo vaccination assays demonstrate that the anti-CTLA-4 response in our models is linked to increased tumor immunogenicity. However, the number of non-synonymous mutations and antigen presentation functionality do not correlate with ICB efficacy. Tumor infiltration by T cells was assessed by CD3 and FoxP3 immunostaining and gene expression analysis. Although clear differential gene expression profiles are noted among the four models and in those tumors responding to the treatment, we unexpectedly found that “hot” melanomas (e.g., showing upregulation of inflammatory pathways and high T-cell infiltration) do not necessarily predict ICB efficacy. These results suggest that additional mechanisms could help determine the response or intrinsic resistance to anti-CTLA-4 and open new avenues for future research and treatment. Overall, our study offers four genetically and phenotypically distinct mouse models representing diverse human melanoma subtypes as powerful tools for the mechanistic study of the response to immunotherapies in melanoma. Citation Format: Corinne Rauck, Eva Perez-Guijarro, Zoe W. Ohler, Rajaa El E. Meskini, Howard Yang, Suman Vodnala, Cari Graff-Cherry, Sung Chin, Anyen Fon, Helen Michael, Maxwell Lee, Terry Van Dyke, Shyam Sharan, Glenn Merlino, Chi-Ping Day. Developing a preclinical immunotherapy platform using syngeneic mouse models of human melanoma [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 5678.

Melanoma is the deadliest form of skin cancer due to the lack of widely effective therapies for advanced disease. Recently FDA-approved immunotherapies, such as immune checkpoint blockade (ICB) by CTLA-4 and PD-1/PD-L1 antibodies, provide unprecedent durable responses but in less than 40% of late stage melanoma patients. While high mutational loads characteristic of responsive tumors has not shown predictive value of patient outcome, accumulating evidences suggest a key role for neoantigens in the response to ICB. Moreover, new T-cell transfer- and vaccine-based therapeutic strategies highlighted the relevance of an efficient identification and prioritization of highly immunogenic neoantigens to improve immunotherapies. However, mechanistic studies are not possible in humans, and the development of adequate predictive methods is still the center of intense debate in the field. Here we use two genetically engineered melanoma mouse models exhibiting distinct response to ICB. Melanomas induced by neonatal ultraviolet radiation (UV) in a HGF-transgenic mouse (HGF-tg) showed high sensitivity to anti-CTLA-4, whereas UV-induced melanomas in HGF-tg; BrafV600E; Cdkn2a+/- mouse (Braf/HGF) did not respond. We hypothesized these models will allow us to identify the neoantigen features required for ICB response including type, expression levels and allele frequency patterns. High tumor immunogenicity, assessed by in vivo vaccination assays, as well as increased T-cell infiltration upon anti-CTLA-4 treatment were correlated to greater response. Moreover, exome and RNA sequencing analyses revealed similar mutational and neoantigen load in both models, albeit with no common expressed mutations. Notably, both models expressed similar levels of antigen presentation related genes (e.g. B2m, H2-Kd, Tap1) suggesting that specific neoantigens in HGF-tg melanoma cells may contribute to their sensitivity to anti-CTLA-4. To test this, we predicted MHC-I/-II binding of HGF-tg melanoma mutated epitopes in silico and generated a “neo-epitope” library. Importantly, the expression and allele frequency of most of selected mutations were decreased in anti-CTLA-4 responder HGF melanomas. The “neo-epitope” library was transduced into Braf/HGF non-responder cells and future studies will identify the neo-epitopes lost upon ICB, representing determinants of therapeutic success. Additionally, in vivo vaccination assays using synthetic mutant peptides will be performed to validate each neoantigen candidate. We anticipate that our studies will provide insight into the role that neoantigens play in melanoma immunotherapy responses. (EPG and CPD contribute to this abstract equally). Citation Format: Eva Perez Guijarro, Chi-Ping Day, Zoe W. Ohler, Rajaa El Meskini, Howard Yang, Suman Vodnala, Cari Graff-Cherry, Sung Chin, Anyen Fon, Helen Michael, Maxwell Lee, Terry Van Dyke, Shyam Sharan, Glenn Merlino. Functional characterization of neoantigens determining immune checkpoint blockade response in mouse models of human melanoma [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 5720.

Immune checkpoint blockers (ICBs) have rendered unprecedented, durable responses in metastatic melanoma, but the heterogeneous response among patients continues to be the major obstacle for their therapeutic development. It is generally hypothesized that neoantigens derived from mutated genes are involved in tumor response to ICBs, since the latter is correlated witt mutational loads of tumors. However, direct experimental evidence showing that threshold quantity or specific properties of neoantigens drive ICB response are mostly lacking. To identify and characterize neoantigens implicated in ICB response, we have generated two UV-induced melanoma models based on the BrafV600E/Pten-knockout (Braf/PKO) and Hgf-transgenic (Hgf-tg) mouse, which displayed intrinsic resistance and high sensitivity to an anti-CTLA-4 antibody, respectively. Exome sequencing identified 216 and 291 non-synonymous mutations in the Braf/PKO and UV-Hgf melanoma cell lines, respectively. By RNA sequencing, 74 (34%) and 121 (42%) of these mutated genes were found to be expressed in each model, respectively, and there were no overlapping mutations between them. The mutations found in the “sensitive” UV-Hgf melanoma were analyzed in silico for their binding affinity to MHC-I and/or MHC-II, thus characterizing putative neoantigens. A “neo-epitope” library was generated by cloning the DNA sequences flanking non-synonymous mutations in frame with the eGFP gene in a lentiviral vector. We further showed that such eGFP-fused epitopes can be presented by the cells to induce specific T cell responses. The library will be transduced into the “resistant” Braf/PKO melanoma, which will be treated with anti-CTLA-4 in mice to identify the neoantigens required for the response. To prevent immunity against eGFP expressed by tumors, the library-transduced melanoma cells will be transplanted into the eGFP-tolerant “glowing head mice”. The results will be used to determine if one, or more, of our candidate neo-epitopes can induce a response to anti-CTLA-4. We will also analyze if the response to this ICB is an epitope-specific reaction or require multiple epitopes, which will help to identify resistance mechanisms. We anticipate that our results will provide insight into the role of neoantigens in ICB response. Moreover, our models will serve as a platform to study the specific contribution and predictive value of neoantigens for melanoma response to immunotherapy, which could help improve therapeutic strategies involving ICBs. Citation Format: Chi-Ping Day, Eva Perez-Guijarro, Rajaa El Meskini, Zoe Weaver Ohler, Maxwell Lee, Howard Yang, Suman Vodnala, Shyam Sharan, Glenn Merlino. Identification of neo-antigens driving melanoma response to immune checkpoint blockers via in vivo screening [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 2623. doi:10.1158/1538-7445.AM2017-2623

Myeloid derived suppressor cells (MDSCs) are increased in tumor bearing condition, and elicit immunosuppression via type 2 polarization. We have previously reported that TGF-β signaling in MDSCs is essential for tumor metastasis. TGFβR2, a receptor essential for TGF-β signaling, is elevated in MDSCs. Deletion of Tgfbr2 in MDSCs, the gene encoding TGFβR2, significantly decreased tumor metastasis. However, it is unclear how TGFβR2 is regulated in MDSCs. We identify microRNA-130a and -145, which directly target Tgfbr2 in MDSCs and inhibit the expression of TGF-β receptor II (TβRII). Both miRs levels were lower in MDSCs from advanced tumor bearing mice correlating to increased TβRII level. Overexpression of miR-130a or -145 in MDSCs shift their immunological phenotype from type 2 to type 1 polarization. Moreover, tumor metastasis was significantly suppressed in myeloid specific miR-130 transgenic mice. In pre-clinical mouse models, the number of lung metastasis was significantly decreased when mice were treated with combination therapy of miR-130a or -145 with paclitaxel when compared with miR-130a, -145, or paclitaxel alone. We proposed that reprogramming of MDSCs by miR-130a and -145 could be a novel strategy in therapeutic treatment for breast cancer metastasis. Citation Format: Hiroki Ishii, Suman K. Vodnala, Bhagelu R. Achyut, Christine M. Hollander, Ashish Lal, Li Yang. miR-130a and -145 reprogram myeloid derived suppressor cells and enhance anti-tumor immunity [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 2944. doi:10.1158/1538-7445.AM2017-2944

CsgD and cyclic-3′,5′-di-guanylate are key regulators of biofilm formation in Salmonella enterica serovar Typhimurium. Our results show that polynucleotide phosphorylase and NlpI oppositely altered expression of CsgD. Polynucleotide phosphorylase and NlpI also had opposite effects on the expression of yjcC , which codes for a cyclic-3′,5′-di-guanylate phosphodiesterase affecting CsgD expression.

Trypanosoma brucei is the causing agent of African trypanosomiasis. These parasites possess a unique thiol redox system required for DNA synthesis and defense against oxidative stress. It includes trypanothione and trypanothione reductase (TryR) instead of the thioredoxin and glutaredoxin systems of mammalian hosts. Here, we show that the benzisothiazolone compound ebsulfur (EbS), a sulfur analogue of ebselen, is a potent inhibitor of T. brucei growth with a favorable selectivity index over mammalian cells. EbS inhibited the TryR activity and decreased non-protein thiol levels in cultured parasites. The inhibition of TryR by EbS was irreversible and NADPH-dependent. EbS formed a complex with TryR and caused oxidation and inactivation of the enzyme. EbS was more toxic for T. brucei than for Trypanosoma cruzi, probably due to lower levels of TryR and trypanothione in T. brucei. Furthermore, inhibition of TryR produced high intracellular reactive oxygen species. Hydrogen peroxide, known to be constitutively high in T. brucei, enhanced the EbS inhibition of TryR. The elevation of reactive oxygen species production in parasites caused by EbS induced a programmed cell death. Soluble EbS analogues were synthesized and cured T. brucei brucei infection in mice when used together with nifurtimox. Altogether, EbS and EbS analogues disrupt the trypanothione system, hampering the defense against oxidative stress. Thus, EbS is a promising lead for development of drugs against African trypanosomiasis.

Host immune surveillance as a mechanism to promote tumor growth or to suppress tumor development is well studied. Less is known how immuno-selective pressure in the form of immunotherapies can instruct the immune system during this process. We hypothesized that the application of a T cell-dependent selection pressure in the form of a whole tumor vaccine would alter the immunogenic architecture in our transplantable murine melanoma model SB-3123. We generated an escape variant to test this hypothesis. We performed whole-exome and RNA-sequencing of the tumor line SB-3123 and two fresh tumors generated by subcutaneous implantation of SB-3123 to identify mutations and to assess levels of expression. There were 349 mutations shared between the tumor line and fresh tumors and less than 5 mutations were unique to each sample. We then investigated whether tumors that had undergone an additional round of host immunoediting would acquire different mutations. To test this, we generated a re-derived tumor cell line from a fresh SB-3123 tumor. Remarkably, the re-derived tissue culture line retained 333 mutations in common with the prior samples and produced only 2 unique mutations. To test the immunogenicity of SB-3123, we vaccinated mice with irradiated SB-3123 and administered a live tumor challenge two weeks after vaccination. This resulted in a profound vaccination response with mice being completely protected from tumor for over 200 days. However, one vaccinated mouse developed a recrudesced tumor at the site of implantation 40 days after challenge and a tumor line was created (SB-3123-esc). We asked whether SB-3123-esc represented an escape variant by immunizing mice with SB-3123 and then challenging with either SB-3123 or SB-3123-esc. Vaccination with SB-3123 did not protect against SB-3123-esc. We also performed sequencing on SB-3123-esc and discovered that 441 nonsynonymous mutations were common to both tumor lines while 80 were unique to SB-3123 and 40 to SB-3123-esc. Furthermore, we were able to determine that SB-3123-esc lost 2 immunogenic neoantigens but had acquired 5. In addition, we identified a murine T cell receptor with specific reactivity for the escape variant but not the parental tumor. Our results demonstrate that the expressed mutations of SB-3123 tumor did not change, regardless of whether we sequenced the tumor line, fresh tumors, or a re-derived tumor cell line. However, the escape tumor had new unique mutations and at least one of these was demonstrably immunogenic; we were able to develop a murine T cell receptor with specific reactivity for the escape variant but not the parental tumor. In conclusion, we posit that these observations offer a proof of concept that the process of cancer immune evasion can create new opportunities to treat escape tumors with T cell based therapies. Citation Format: Jenny H. Pan, Suman Vodnala, Robert L. Eil, Zhiya Yu, Jared Gartner, David Clever, Rahul Roychoudhuri, Shashank J. Patel, Christopher A. Klebanoff, Madhu Sukumar, Tori Yamamoto, Nicholas P. Restifo. Immunotherapy generates selective pressure to create an escape tumor with increased susceptibility to treatment with T cell based therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4034.

The aim of this study was to synthesize a series of mono-, di- and trisubstituted derivatives of the human African trypanosomiasis drug eflornithine (alpha-difluoromethylornithine, DMFO, CAS 70052-12-9) to determine their partition coefficients, and to assess whether they deliver the parent drug in the plasma. If increased plasma concentrations of eflornithine could be achieved in this way, an oral dosage form would be possible. The derivatives, nine in total, were successfully synthesized by multi-step derivatisation of eflornithine on either its alpha-carboxylic or/and alpha-amino or/and delta-amino groups by either esterification or/and amidation or/and carbamylation, and their structures confirmed by NMR and MS spectroscopy. The majority of derivatives were more lipophilic than eflornithine with log D values in phosphate buffer solution (pH 7.4) ranging from -1.34 to 1.59 (vs. -0.98 for eflornithine). The in vivo absorption after oral administration to Sprague-Dawley rats showed that no derivative delivered eflornithine in the plasma, indicating that the derivatives were either not absorbed from the gastrointestinal tract or not metabolized to the parent drug. Two of the monosubstituted activities were toxic for T. brucei blood stream forms.

Background There is an urgent need to substitute the highly toxic compounds still in use for treatment of the encephalitic stage of human African trypanosomiasis (HAT). We here assessed the treatment with the doublet cordycepin and the deaminase inhibitor deoxycoformycin for this stage of infection with Trypanosoma brucei (T.b.). Methodology/Principal Findings Cordycepin was selected as the most efficient drug from a direct parasite viability screening of a compound library of nucleoside analogues. The minimal number of doses and concentrations of the drugs effective for treatment of T.b. brucei infections in mice were determined. Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment. The doublet was effective for treatment of late stage experimental infections with human pathogenic T.b. rhodesiense and T.b. gambiense isolates. Late stage infection treatment diminished the levels of inflammatory cytokines in brains of infected mice. Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites. T.b. brucei strains developed resistance to cordycepin after culture with increasing concentrations of the compound. However, cordycepin-resistant parasites showed diminished virulence and were not cross-resistant to other drugs used for treatment of HAT, i.e. pentamidine, suramin and melarsoprol. Although resistant parasites were mutated in the gene coding for P2 nucleoside adenosine transporter, P2 knockout trypanosomes showed no altered resistance to cordycepin, indicating that absence of the P2 transporter is not sufficient to render the trypanosomes resistant to the drug. Conclusions/Significance Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT., Author Summary There is an urgent need to substitute the highly toxic arsenic compounds still in use for treatment of the encephalitic stage of African trypanosomiasis, a disease caused by infection with Trypanosoma brucei. We exploited the inability of trypanosomes to engage in de novo purine synthesis as a therapeutic target. Cordycepin was selected from a trypanocidal screen of a 2200-compound library. When administered together with the adenosine deaminase inhibitor deoxycoformycin, cordycepin cured mice inoculated with the human pathogenic subspecies T. brucei rhodesiense or T. brucei gambiense even after parasites had penetrated into the brain. Successful treatment was achieved by intraperitoneal, oral or subcutaneous administration of the compounds. Treatment with the doublet also diminished infection-induced cerebral inflammation. Cordycepin induced programmed cell death of the parasites. Although parasites grown in vitro with low doses of cordycepin gradually developed resistance, the resistant parasites lost virulence and showed no cross-resistance to trypanocidal drugs in clinical use. Our data strongly support testing cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.

Monitoring Trypanosoma spread using real-time imaging in vivo provides a fast method to evaluate parasite distribution especially in immunoprivileged locations. Here, we generated monomorphic and pleomorphic recombinant Trypanosoma brucei expressing the Renilla luciferase. In vitro luciferase activity measurements confirmed the uptake of the coelenterazine substrate by live parasites and light emission. We further validated the use of Renilla luciferase-tagged trypanosomes for real-time bioluminescent in vivo analysis. Interestingly, a preferential testis tropism was observed with both the monomorphic and pleomorphic recombinants. This is of importance when considering trypanocidal drug development, since parasites might be protected from many drugs by the blood-testis barrier. This hypothesis was supported by our final study of the efficacy of treatment with trypanocidal drugs in T. brucei-infected mice. We showed that parasites located in the testis, as compared to those located in the abdominal cavity, were not readily cleared by the drugs., Author Summary Human African trypanosomiasis or sleeping sickness, caused by two subspecies of Trypanosoma brucei, is endemic in Subsaharan Africa. There is no vaccine and the currently used drugs are toxic and can cause severe side effects and even death. At present, we do not know how and when parasites can leave the blood and penetrate into organs (especially the brain). Such knowledge will be very helpful to develop and validate new drugs that can clear the parasite from both the blood and the tissues. In this study, we developed a novel technique allowing us to track the parasites in a live animal by the use of light signals. By following the luminescent parasites in the mouse we showed that, interestingly, the organisms migrate very early in infection to the testes. Here, they may be protected from the immune system and from drugs. Indeed when treating the mice with a sub-optimal dose of medicine, the parasites in this location were not cleared and subsequently could cause a reinvasion into the blood of the host.

Meeting abstracts Host immunosurveillance as a mechanism to promote tumor growth or to suppress tumor development is well studied. It is less known how immuno-selective pressure in the form of immunotherapies can instruct the immune system during this process. We hypothesized that the application

Tumor derived soluble factors result in accumulation of Gr-1+CD11b+ immature myeloid cells at tumor site and premetastatic organ. These cells are known to promote tumor progression and metastasis. Gr-1+CD11b+ immature myeloid cells and tumor-associated macrophages display a type 2 phenotype (M2) and produce immune suppressive cytokines including TGFβ. We have previously shown that immature myeloid cells are vicious producers of TGFβ ligand and express high levels of TGFβR2 receptor for eliciting immune suppressive function. Specific deletion of TGFβR2 in myeloid cells resulted in increased antitumor immunity and decreased metastasis in the lungs of cancer mouse models. microRNAs (miRs) are abundant class of small non-coding RNAs that have recently emerged as powerful epigenetic regulators of gene expression in pathophysiological conditions. One of the critical properties of miR is to target a large number of genes frequently within the context of a network, making them very efficient in regulating distinct biological cell processes. In the current project, we have identified three miRs that target myeloid TGFβR2 associated molecular network including p38 and cAMP pathways, which are critical in type 2 cytokine production. We have successfully transduced bone marrow stem cells with lentivirus expressing these miRs driven by a myeloid specific promoter CD11b. Transplantation of these modified bone marrow into lethally irradiated mice decreased breast cancer metastasis. The underlying mechanisms involve the reduction in type 2 cytokines such as IL-4, IL-10 and IL-13 and activation of IFN producing CD8 cytotoxic lymphocytes. Interestingly, the miRNAs are also differentially regulated by TGFβ signaling in TGFβR2+/+ and TGFβR2−/− myeloid cells, suggesting a feedback loop between TGFβ and miRNA. These results demonstrate that the candidate miRNA can reprogram tumor-associated myeloid cells by altering the cytokine milieu and enhance host antitumor immunity. Our work points out that endogenous miRNA targeting myeloid TGFβ signaling and its molecular network provides an important therapeutic option. Note: This abstract was not presented at the meeting. Citation Format: Suman K. Vodnala. MicroRNA-mediated reprogramming of myeloid cells by targeting TGFβ signaling and its associated molecular network. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3128. doi:10.1158/1538-7445.AM2015-3128