Inhibition of choline metabolism in an angioimmunoblastic T-cell lymphoma preclinical model reveals a new metabolic vulnerability as possible target for treatment.

BACKGROUND: Angioimmunoblastic T-cell lymphoma (AITL) is a malignancy with very poor survival outcome, in urgent need of more specific therapeutic strategies. The drivers of malignancy in this disease are CD4+ follicular helper T cells (Tfh). The metabolism of these malignant Tfh cells was not yet elucidated. Therefore, we decided to identify their metabolic requirements with the objective to propose a novel therapeutic option. METHODS: To reveal the prominent metabolic pathways used by the AITL lymphoma cells, we relied on metabolomic and proteomic analysis of murine AITL (mAITL) T cells isolated from our established mAITL model. We confirmed these results using AITL patient and healthy T cell expression data. RESULTS: Strikingly, the mAITL Tfh cells were highly dependent on the second branch of the Kennedy pathway, the choline lipid pathway, responsible for the production of the major membrane constituent phosphatidylcholine. Moreover, gene expression data from Tfh cells isolated from AITL patient tumors, confirmed the upregulation of the choline lipid pathway. Several enzymes involved in this pathway such as choline kinase, catalyzing the first step in the phosphatidylcholine pathway, are upregulated in multiple tumors other than AITL. Here we showed that treatment of our mAITL preclinical mouse model with a fatty acid oxydation inhibitor, significantly increased their survival and even reverted the exhausted CD8 T cells in the tumor into potent cytotoxic anti-tumor cells. Specific inhibition of Chokα confirmed the importance of the phosphatidylcholine production pathway in neoplastic CD4 + T cells, nearly eradicating mAITL Tfh cells from the tumors. Finally, the same inhibitor induced in human AITL lymphoma biopsies cell death of the majority of the hAITL PD-1high neoplastic cells. CONCLUSION: Our results suggest that interfering with choline metabolism in AITL reveals a specific metabolic vulnerability and might represent a new therapeutic strategy for these patients.

Cytidine Deaminase Resolves Replicative Stress and Protects Pancreatic Cancer from DNA-Targeting Drugs.

Cytidine deaminase (CDA) functions in the pyrimidine salvage pathway for DNA and RNA syntheses and has been shown to protect cancer cells from deoxycytidine-based chemotherapies. In this study, we observed that CDA was overexpressed in pancreatic adenocarcinoma from patients at baseline and was essential for experimental tumor growth. Mechanistic investigations revealed that CDA localized to replication forks where it increased replication speed, improved replication fork restart efficiency, reduced endogenous replication stress, minimized DNA breaks, and regulated genetic stability during DNA replication. In cellular pancreatic cancer models, high CDA expression correlated with resistance to DNA-damaging agents. Silencing CDA in patient-derived primary cultures in vitro and in orthotopic xenografts in vivo increased replication stress and sensitized pancreatic adenocarcinoma cells to oxaliplatin. This study sheds light on the role of CDA in pancreatic adenocarcinoma, offering insights into how this tumor type modulates replication stress. These findings suggest that CDA expression could potentially predict therapeutic efficacy and that targeting CDA induces intolerable levels of replication stress in cancer cells, particularly when combined with DNA-targeted therapies. SIGNIFICANCE: Cytidine deaminase reduces replication stress and regulates DNA replication to confer resistance to DNA-damaging drugs in pancreatic cancer, unveiling a molecular vulnerability that could enhance treatment response.

Patient-derived lymphoma spheroids integrating immune tumor microenvironment as preclinical follicular lymphoma models for personalized medicine.

BACKGROUND: Follicular lymphoma (FL), the most common indolent non-Hodgkin's Lymphoma, is a heterogeneous disease and a paradigm of the contribution of immune tumor microenvironment to disease onset, progression, and therapy resistance. Patient-derived models are scarce and fail to reproduce immune phenotypes and therapeutic responses. METHODS: To capture disease heterogeneity and microenvironment cues, we developed a patient-derived lymphoma spheroid (FL-PDLS) model culturing FL cells from lymph nodes (LN) with an optimized cytokine cocktail that mimics LN stimuli and maintains tumor cell viability. RESULTS: FL-PDLS, mainly composed of tumor B cells (60% on average) and autologous T cells (13% CD4 and 3% CD8 on average, respectively), rapidly organizes into patient-specific three-dimensional (3D) structures of three different morphotypes according to 3D imaging analysis. RNAseq analysis indicates that FL-PDLS reproduces FL hallmarks with the overexpression of cell cycle, BCR, or mTOR signaling related gene sets. FL-PDLS also recapitulates the exhausted immune phenotype typical of FL-LN, including expression of BTLA, TIGIT, PD-1, TIM-3, CD39 and CD73 on CD3+ T cells. These features render FL-PDLS an amenable system for immunotherapy testing. With this aim, we demonstrate that the combination of obinutuzumab (anti-CD20) and nivolumab (anti-PD1) reduces tumor load in a significant proportion of FL-PDLS. Interestingly, B cell depletion inversely correlates with the percentage of CD8+ cells positive for PD-1 and TIM-3. CONCLUSIONS: In summary, FL-PDLS is a robust patient-derived 3D system that can be used as a tool to mimic FL pathology and to test novel immunotherapeutic approaches in a context of personalized medicine.

An agent-based model of monocyte differentiation into tumour-associated macrophages in chronic lymphocytic leukemia.

Monocyte-derived macrophages help maintain tissue homeostasis and defend the organism against pathogens. In tumors, recent studies have uncovered complex macrophage populations, including tumor-associated macrophages, which support tumorigenesis through cancer hallmarks such as immunosuppression, angiogenesis, or matrix remodeling. In the case of chronic lymphocytic leukemia, these macrophages are known as nurse-like cells (NLCs) and they protect leukemic cells from spontaneous apoptosis, contributing to their chemoresistance. We propose an agent-based model of monocyte differentiation into NLCs upon contact with leukemic B cells in vitro. We performed patient-specific model optimization using cultures of peripheral blood mononuclear cells from patients. Using our model, we were able to reproduce the temporal survival dynamics of cancer cells in a patient-specific manner and to identify patient groups related to distinct macrophage phenotypes. Our results show a potentially important role of phagocytosis in the polarization process of NLCs and in promoting cancer cells' enhanced survival.

The proteome and transcriptome of stress granules and P bodies during human T lymphocyte activation.

Stress granules (SGs) and processing bodies (PBs) are membraneless cytoplasmic assemblies regulating mRNAs under environmental stress such as viral infections, neurological disorders, or cancer. Upon antigen stimulation, T lymphocytes mediate their immune functions under regulatory mechanisms involving SGs and PBs. However, the impact of T cell activation on such complexes in terms of formation, constitution, and relationship remains unknown. Here, by combining proteomic, transcriptomic, and immunofluorescence approaches, we simultaneously characterized the SGs and PBs from primary human T lymphocytes pre and post stimulation. The identification of the proteomes and transcriptomes of SGs and PBs indicate an unanticipated molecular and functional complementarity. Notwithstanding, these granules keep distinct spatial organizations and abilities to interact with mRNAs. This comprehensive characterization of the RNP granule proteomic and transcriptomic landscapes provides a unique resource for future investigations on SGs and PBs in T lymphocytes.

Single-cell spatial explorer: easy exploration of spatial and multimodal transcriptomics.

BACKGROUND: The development of single-cell technologies yields large datasets of information as diverse and multimodal as transcriptomes, immunophenotypes, and spatial position from tissue sections in the so-called 'spatial transcriptomics'. Currently however, user-friendly, powerful, and free algorithmic tools for straightforward analysis of spatial transcriptomic datasets are scarce. RESULTS: Here, we introduce Single-Cell Spatial Explorer, an open-source software for multimodal exploration of spatial transcriptomics, examplified with 9 human and murine tissues datasets from 4 different technologies. CONCLUSIONS: Single-Cell Spatial Explorer is a very powerful, versatile, and interoperable tool for spatial transcriptomics analysis.

Cáncer de hígado: ¿Antigua o nueva amenaza para los jóvenes de países en vía de desarrollo?

Entre las neoplasias malignas más frecuentes del mundo, el carcinoma hepatocelular (CHC) es la segunda causa de muerte relacionada con el cáncer (1). Su incidencia se ha duplicado durante las dos últimas décadas y la mayor carga se produce en los países de ingresos bajos y medianos. Los tumores hepáticos primarios malignos suelen describirse como una patología que afecta principalmente a hombres mayores de 40 años con un hígado cirrótico; rara vez se han registrado en personas más jóvenes y normalmente, en menores de 40, lo más común es el hepatoblastoma

Baseline SUVmax is related to tumor cell proliferation and patient outcome in follicular lymphoma.

Follicular lymphoma (FL) is the most common indolent lymphoma. Despite the clear benefit of CD20-based therapy, a subset of FL patients still progress to aggressive lymphoma. Thus, identifying early biomarkers that incorporate PET metrics could be helpful to identify patients with a high risk of treatment failure with Rituximab. We retrospectively included a total of 132 untreated FL patients separated into training and validation cohorts. Optimal threshold of baseline SUVmax was first determined in the training cohort (n=48) to predict progression-free survival (PFS). The PET results were investigated along with the tumor and immune microenvironment, which were determined by immunochemistry and transcriptome studies involving gene set enrichment analyses and immune cell deconvolution, together with the tumor mutation profile. We report that baseline SUVmax >14.5 was associated with poorer PFS than baseline SUVmax ≤14.5 (HR=0.28; p=0.00046). Neither immune T-cell infiltration nor immune checkpoint expression were associated with baseline PET metrics. By contrast, FL samples with Ki-67 staining ≥10% showed enrichment of cell cycle/DNA genes (p=0.013) and significantly higher SUVmax values (p=0.007). Despite similar oncogenic pathway alterations in both SUVmax groups of FL samples, 4 out of 5 cases harboring the infrequent FOXO1 transcription factor mutation were seen in FL patients with SUVmax >14.5. Thus, high baseline SUVmax reflects FL tumor proliferation and, together with Ki-67 proliferative index, can be used to identify patients at risk of early relapse with R-chemotherapy.

Single-Cell RNAseq Profiling of Human γδ T Lymphocytes in Virus-Related Cancers and COVID-19 Disease.

The detailed characterization of human γδ T lymphocyte differentiation at the single-cell transcriptomic (scRNAseq) level in tumors and patients with coronavirus disease 2019 (COVID-19) requires both a reference differentiation trajectory of γδ T cells and a robust mapping method for additional γδ T lymphocytes. Here, we incepted such a method to characterize thousands of γδ T lymphocytes from (n = 95) patients with cancer or adult and pediatric COVID-19 disease. We found that cancer patients with human papillomavirus-positive head and neck squamous cell carcinoma and Epstein-Barr virus-positive Hodgkin's lymphoma have γδ tumor-infiltrating T lymphocytes that are more prone to recirculate from the tumor and avoid exhaustion. In COVID-19, both TCRVγ9 and TCRVγnon9 subsets of γδ T lymphocytes relocalize from peripheral blood mononuclear cells (PBMC) to the infected lung tissue, where their advanced differentiation, tissue residency, and exhaustion reflect T cell activation. Although severe COVID-19 disease increases both recruitment and exhaustion of γδ T lymphocytes in infected lung lesions but not blood, the anti-IL6R therapy with Tocilizumab promotes γδ T lymphocyte differentiation in patients with COVID-19. PBMC from pediatric patients with acute COVID-19 disease display similar γδ T cell lymphopenia to that seen in adult patients. However, blood γδ T cells from children with the COVID-19-related multisystem inflammatory syndrome are not lymphodepleted, but they are differentiated as in healthy PBMC. These findings suggest that some virus-induced memory γδ T lymphocytes durably persist in the blood of adults and could subsequently infiltrate and recirculate in tumors.

Phased differentiation of γδ T and T CD8 tumor-infiltrating lymphocytes revealed by single-cell transcriptomics of human cancers.

γδ T lymphocytes diverge from conventional T CD8 lymphocytes for ontogeny, homing, and antigen specificity, but whether their differentiation in tumors also deviates was unknown. Using innovative analyses of our original and ~150 published single-cell RNA sequencing datasets validated by phenotyping of human tumors and murine models, here we present the first high-resolution view of human γδ T cell differentiation in cancer. While γδ T lymphocytes prominently encompass TCRVγ9 cells more differentiated than T CD8 in healthy donor's blood, a different scenario is unveiled in tumors. Solid tumors and lymphomas are infiltrated by a majority of TCRVγnon9 γδ T cells which are quantitatively correlated and remarkably aligned with T CD8 for differentiation, exhaustion, gene expression profile, and response to immune checkpoint therapy. This cancer-wide association is critical for developing cancer immunotherapies.

Self-activation of Vγ9Vδ2 T cells by exogenous phosphoantigens involves TCR and butyrophilins.

The high cytotoxic activity of Vγ9Vδ2 T lymphocytes against tumor cells makes them useful candidates in anticancer therapies. However, the molecular mechanism of their activation by phosphoantigens (PAgs) is not completely known. Many studies have depicted the mechanism of Vγ9Vδ2 T-cell activation by PAg-sensed accessory cells, such as immune presenting cells or tumor cells. In this study, we demonstrated that pure resting Vγ9Vδ2 T lymphocytes can self-activate through exogenous PAgs, involving their TCR and the butyrophilins BTN3A1 and BTN2A1. This is the first time that these three molecules, concurrently expressed at the plasma membrane of Vγ9Vδ2 T cells, have been shown to be involved together on the same and unique T cell during PAg activation. Moreover, the use of probucol to stimulate the inhibition of this self-activation prompted us to propose that ABCA-1 could be implicated in the transfer of exogenous PAgs inside Vγ9Vδ2 T cells before activating them through membrane clusters formed by γ9TCR, BTN3A1 and BTN2A1. The self-activation of Vγ9Vδ2 T cells, which leads to self-killing, can therefore participate in the failure of γδ T cell-based therapies with exogenous PAgs and should be taken into account.

Cancer cell adaptability: turning ribonucleoprotein granules into targets.

Stress granules (SGs) and processing bodies (P-bodies) are membraneless cytoplasmic condensates of ribonucleoproteins (RNPs). They both regulate RNA fate under physiological and pathological conditions, and are thereby involved in the regulation and maintenance of cellular integrity. During tumorigenesis, cancer cells use these granules to thrive, to adapt to the harsh conditions of the tumor microenvironment (TME), and to protect themselves from anticancer treatments. This ability to provide multiple outcomes not only makes RNP granules promising targets for cancer therapy but also emphasizes the need for more knowledge about the biology of these granules to achieve clinical use. In this review we focus on the role of RNP granules in cancer, and on how their composition and regulation might be used to elaborate therapeutic strategies.

3D Model Characterization by 2D and 3D Imaging in t(14;18)-Positive B-NHL: Perspectives for In Vitro Drug Screens in Follicular Lymphoma.

Follicular lymphoma (FL) is an indolent B cell lymphoproliferative disorder of transformed follicular center B cells, which accounts for 20-30 percent of all non-Hodgkin lymphoma (NHL) cases. Great advances have been made to identify the most relevant targets for precision therapy. However, no relevant models for in vitro studies have been developed or characterized in depth. To this purpose, we generated a 3D cell model from t(14;18)-positive B-NHL cell lines cultured in ultra-low attachment 96-well plates. Morphological features and cell growth behavior were evaluated by classical microscopy (2D imaging) and response to treatment with different drugs was evaluated by a high-content analysis system to determine the robustness of the model. We show that the ultra-low attachment (ULA) method allows the development of regular, spherical and viable ULA-multicellular aggregates of lymphoma cells (MALC). However, discrepancies in the results obtained after 2D imaging analyses on drug-treated ULA-MALC prompted us to develop 3D imaging and specific analyses. We show by using light sheet microscopy and specifically developed 3D imaging algorithms that 3D imaging and dedicated analyses are necessary to characterize morphological properties of 3D models and drug effects. This study proposes a new method, but also imaging tools and informatic solutions, developed for FL necessary for future preclinical studies.

Lymphoma Heterogeneity Unraveled by Single-Cell Transcriptomics.

High-definition transcriptomic studies through single-cell RNA sequencing (scRNA-Seq) have revealed the heterogeneity and functionality of the various microenvironments across numerous solid tumors. Those pioneer studies have highlighted different cellular signatures correlated with clinical response to immune checkpoint inhibitors. scRNA-Seq offers also a unique opportunity to unravel the intimate heterogeneity of the ecosystems across different lymphoma entities. In this review, we will first cover the basics and future developments of the technology, and we will discuss its input in the field of translational lymphoma research, from determination of cell-of-origin and functional diversity, to monitoring of anti-cancer targeted drugs response and toxicities, and how new improvements in both data collection and interpretation will further foster precision medicine in the upcoming years.

Global DNA hypermethylation pattern and unique gene expression signature in liver cancer from patients with Indigenous American ancestry.

Hepatocellular carcinoma (HCC) usually afflicts individuals in their maturity after a protracted liver disease. Contrasting with this pattern, the age structure of HCC in Andean people displays a bimodal distribution with half of the patients developing HCC in adolescence and early adulthood. To deepen our understanding of the molecular determinants of the disease in this population, we conducted an integrative analysis of gene expression and DNA methylation in HCC developed by 74 Peruvian patients, including 39 adolescents and young adults. While genome-wide hypomethylation is considered as a paradigm in human HCCs, our analysis revealed that Peruvian tumors are associated with a global DNA hypermethylation. Moreover, pathway enrichment analysis of transcriptome data characterized an original combination of signatures. Peruvian HCC forgoes canonical activations of IGF2, Notch, Ras/MAPK, and TGF-ß signals to depend instead on Hippo/YAP1, MYC, and Wnt/ß-catenin pathways. These signatures delineate a homogeneous subtype of liver tumors at the interface of the proliferative and non-proliferative classes of HCCs. Remarkably, the development of this HCC subtype occurs in patients with one of the four Native American mitochondrial haplogroups A-D. Finally, integrative characterization revealed that Peruvian HCC is apparently controlled by the PRC2 complex that mediates cell reprogramming with massive DNA methylation modulating gene expression and pinpointed retinoid signaling as a potential target for epigenetic therapy.

Cancer Cells Resistance Shaping by Tumor Infiltrating Myeloid Cells.

Interactions between malignant cells and neighboring stromal and immune cells profoundly shape cancer progression. New forms of therapies targeting these cells have revolutionized the treatment of cancer. However, in order to specifically address each population, it was essential to identify and understand their individual roles in interaction between malignant cells, and the formation of the tumor microenvironment (TME). In this review, we focus on the myeloid cell compartment, a prominent, and heterogeneous group populating TME, which can initially exert an anti-tumoral effect, but with time actively participate in disease progression. Macrophages, dendritic cells, neutrophils, myeloid-derived suppressor cells, mast cells, eosinophils, and basophils act alone or in concert to shape tumor cells resistance through cellular interaction and/or release of soluble factors favoring survival, proliferation, and migration of tumor cells, but also immune-escape and therapy resistance.

IL-10 Rescues CLL Survival through Repolarization of Inflammatory Nurse-like Cells.

Tumor-associated macrophages (TAMs) in chronic lymphocytic leukemia (CLL) are also called nurse-like cells (NLC), and confer survival signals through the release of soluble factors and cellular contacts. While in most patient samples the presence of NLC in co-cultures guarantees high viability of leukemic cells in vitro, in some cases this protective effect is absent. These macrophages are characterized by an "M1-like phenotype". We show here that their reprogramming towards an M2-like phenotype (tumor-supportive) with IL-10 leads to an increase in leukemic cell survival. Inflammatory cytokines, such as TNF, are also able to depolarize M2-type protective NLC (decreasing CLL cell viability), an effect which is countered by IL-10 or blocking antibodies. Interestingly, both IL-10 and TNF are implied in the pathophysiology of CLL and their elevated level is associated with bad prognosis. We propose that the molecular balance between these two cytokines in CLL niches plays an important role in the maintenance of the protective phenotype of NLCs, and therefore in the survival of CLL cells.

Mitochondrial inhibitors circumvent adaptive resistance to venetoclax and cytarabine combination therapy in acute myeloid leukemia.

Therapy resistance represents a major clinical challenge in acute myeloid leukemia (AML). Here we define a 'MitoScore' signature, which identifies high mitochondrial oxidative phosphorylation in vivo and in patients with AML. Primary AML cells with cytarabine (AraC) resistance and a high MitoScore relied on mitochondrial Bcl2 and were highly sensitive to venetoclax (VEN) + AraC (but not to VEN + azacytidine). Single-cell transcriptomics of VEN + AraC-residual cell populations revealed adaptive resistance associated with changes in oxidative phosphorylation, electron transport chain complex and the TP53 pathway. Accordingly, treatment of VEN + AraC-resistant AML cells with electron transport chain complex inhibitors, pyruvate dehydrogenase inhibitors or mitochondrial ClpP protease agonists substantially delayed relapse following VEN + AraC. These findings highlight the central role of mitochondrial adaptation during AML therapy and provide a scientific rationale for alternating VEN + azacytidine with VEN + AraC in patients with a high MitoScore and to target mitochondrial metabolism to enhance the sensitivity of AML cells to currently approved therapies.

PD-1 blockade restores helper activity of tumor-infiltrating, exhausted PD-1hiCD39+ CD4 T cells.

Tumor antigen-specific CD4 T cells accumulate at tumor sites, evoking their involvement in antitumor effector functions in situ. Contrary to CD8 cytotoxic T lymphocyte exhaustion, that of CD4 T cells remains poorly appreciated. Here, using phenotypic, transcriptomic, and functional approaches, we characterized CD4 T cell exhaustion in patients with head and neck, cervical, and ovarian cancer. We identified a CD4 tumor-infiltrating lymphocyte (TIL) population, defined by high PD-1 and CD39 expression, which contained high proportions of cytokine-producing cells, although the quantity of cytokines produced by these cells was low, evoking an exhausted state. Terminal exhaustion of CD4 TILs was instated regardless of TIM-3 expression, suggesting divergence with CD8 T cell exhaustion. scRNA-Seq and further phenotypic analyses uncovered similarities with the CD8 T cell exhaustion program. In particular, PD-1hiCD39+ CD4 TILs expressed the exhaustion transcription factor TOX and the chemokine CXCL13 and were tumor antigen specific. In vitro, PD-1 blockade enhanced CD4 TIL activation, as evidenced by increased CD154 expression and cytokine secretion, leading to improved dendritic cell maturation and consequently higher tumor-specific CD8 T cell proliferation. Our data identify exhausted CD4 TILs as players in responsiveness to immune checkpoint blockade.

Insights on TAM Formation from a Boolean Model of Macrophage Polarization Based on In Vitro Studies.

The tumour microenvironment is the surrounding of a tumour, including blood vessels, fibroblasts, signaling molecules, the extracellular matrix and immune cells, especially neutrophils and monocyte-derived macrophages. In a tumour setting, macrophages encompass a spectrum between a tumour-suppressive (M1) or tumour-promoting (M2) state. The biology of macrophages found in tumours (Tumour Associated Macrophages) remains unclear, but understanding their impact on tumour progression is highly important. In this paper, we perform a comprehensive analysis of a macrophage polarization network, following two lines of enquiry: (i) we reconstruct the macrophage polarization network based on literature, extending it to include important stimuli in a tumour setting, and (ii) we build a dynamical model able to reproduce macrophage polarization in the presence of different stimuli, including the contact with cancer cells. Our simulations recapitulate the documented macrophage phenotypes and their dependencies on specific receptors and transcription factors, while also unravelling the formation of a special type of tumour associated macrophages in an in vitro model of chronic lymphocytic leukaemia. This model constitutes the first step towards elucidating the cross-talk between immune and cancer cells inside tumours, with the ultimate goal of identifying new therapeutic targets that could control the formation of tumour associated macrophages in patients.