Development of NR0B2 as a therapeutic target for the re-education of tumor associated myeloid cells.

Immune checkpoint blockade (ICB) has had limited utility in several solid tumors such as breast cancer, a major cause of cancer-related mortality in women. Therefore, there is considerable interest in alternate strategies to promote an anti-cancer immune response. A paper co-published in this issue describes how NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the inflammasome and reduce the expansion of immune-suppressive regulatory T cells (Treg). Here, we develop NR0B2 as a potential therapeutic target. NR0B2 in tumors is associated with improved survival for several cancer types including breast. Importantly, NR0B2 expression is also prognostic of ICB success. Within breast tumors, NR0B2 expression is inversely associated with FOXP3, a marker of Tregs. While a described agonist (DSHN) had some efficacy, it required high doses and long treatment times. Therefore, we designed and screened several derivatives. A methyl ester derivative (DSHN-OMe) emerged as superior in terms of (1) cellular uptake, (2) ability to regulate expected expression of genes, (3) suppression of Treg expansion using in vitro co-culture systems, and (4) efficacy against the growth of primary and metastatic tumors. This work identifies NR0B2 as a target to re-educate myeloid immune cells and a novel ligand with significant anti-tumor efficacy in preclinical models.

NR0B2 re-educates myeloid immune cells to reduce regulatory T cell expansion and progression of breast and other solid tumors.

Although survival from breast cancer has dramatically increased, many will develop recurrent, metastatic disease. Unfortunately, survival for this stage of disease remains very low. Activating the immune system has incredible promise since it has the potential to be curative. However, immune checkpoint blockade (ICB) which works through T cells has been largely disappointing for metastatic breast cancer. One reason for this is a suppressive myeloid immune compartment that is unaffected by ICB. Cholesterol metabolism and proteins involved in cholesterol homeostasis play important regulatory roles in myeloid cells. Here, we demonstrate that NR0B2, a nuclear receptor involved in negative feedback of cholesterol metabolism, works in several myeloid cell types to impair subsequent expansion of regulatory T cells (Tregs); Tregs being a subset known to be highly immune suppressive and associated with poor therapeutic response. Within myeloid cells, NR0B2 serves to decrease many aspects of the inflammasome, ultimately resulting in decreased IL1ß; IL1ß driving Treg expansion. Importantly, mice lacking NR0B2 exhibit accelerated tumor growth. Thus, NR0B2 represents an important node in myeloid cells dictating ensuing Treg expansion and tumor growth, thereby representing a novel therapeutic target to re-educate these cells, having impact across different solid tumor types. Indeed, a paper co-published in this issue demonstrates the therapeutic utility of targeting NR0B2.

Valrubicin-loaded immunoliposomes for specific vesicle-mediated cell death in the treatment of hematological cancers.

We created valrubicin-loaded immunoliposomes (Val-ILs) using the antitumor prodrug valrubicin, a hydrophobic analog of daunorubicin. Being lipophilic, valrubicin readily incorporated Val-lLs that were loaded with specific antibodies. Val-ILs injected intravenously rapidly reached the bone marrow and spleen, indicating their potential to effectively target cancer cells in these areas. Following the transplantation of human pediatric B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), or acute myeloid leukemia (AML) in immunodeficient NSG mice, we generated patient-derived xenograft (PDX) models, which were treated with Val-ILs loaded with antibodies to target CD19, CD7 or CD33. Only a small amount of valrubicin incorporated into Val-ILs was needed to induce leukemia cell death in vivo, suggesting that this approach could be used to efficiently treat acute leukemia cells. We also demonstrated that Val-ILs could reduce the risk of contamination of CD34+ hematopoietic stem cells by acute leukemia cells during autologous peripheral blood stem cell transplantation, which is a significant advantage for clinical applications. Using EL4 lymphoma cells on immunocompetent C57BL/6 mice, we also highlighted the potential of Val-ILs to target immunosuppressive cell populations in the spleen, which could be valuable in impairing cancer cell expansion, particularly in lymphoma cases. The most efficient Val-ILs were found to be those loaded with CD11b or CD223 antibodies, which, respectively, target the myeloid-derived suppressor cells (MDSC) or the lymphocyte-activation gene 3 (LAG-3 or CD223) on T4 lymphocytes. This study provides a promising preclinical demonstration of the effectiveness and ease of preparation of Val-ILs as a novel nanoparticle technology. In the context of hematological cancers, Val-ILs have the potential to be used as a precise and effective therapy based on targeted vesicle-mediated cell death.

Re-education of myeloid immune cells to reduce regulatory T cell expansion and impede breast cancer progression.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy but has had limited utility in several solid tumors such as breast cancer, a major cause of cancer-related mortality in women. Therefore, there is considerable interest in alternate strategies to promote an anti-cancer immune response. We demonstrate that NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the NLRP3 inflammasome and reduce the expansion of immune-suppressive regulatory T cells (Treg). Loss of NR0B2 increased mammary tumor growth and metastasis. Small molecule agonists, including one developed here, reduced Treg expansion, reduced metastatic growth and improved the efficacy of ICB. This work identifies NR0B2 as a target to re-educate myeloid immune cells providing proof-of-principle that this cholesterol-homeostasis axis may have utility in enhancing ICB.

GTN Enhances Antitumor Effects of Doxorubicin in TNBC by Targeting the Immunosuppressive Activity of PMN-MDSC.

(1) Background: Immunosuppression is a key barrier to effective anti-cancer therapies, particularly in triple-negative breast cancer (TNBC), an aggressive and difficult to treat form of breast cancer. We investigated here whether the combination of doxorubicin, a standard chemotherapy in TNBC with glyceryltrinitrate (GTN), a nitric oxide (NO) donor, could overcome chemotherapy resistance and highlight the mechanisms involved in a mouse model of TNBC. (2) Methods: Balb/C-bearing subcutaneous 4T1 (TNBC) tumors were treated with doxorubicin (8 mg/Kg) and GTN (5 mg/kg) and monitored for tumor growth and tumor-infiltrating immune cells. The effect of treatments on MDSCs reprogramming was investigated ex vivo and in vitro. (3) Results: GTN improved the anti-tumor efficacy of doxorubicin in TNBC tumors. This combination increases the intra-tumor recruitment and activation of CD8+ lymphocytes and dampens the immunosuppressive function of PMN-MDSCs PD-L1low. Mechanistically, in PMN-MDSC, the doxorubicin/GTN combination reduced STAT5 phosphorylation, while GTN +/- doxorubicin induced a ROS-dependent cleavage of STAT5 associated with a decrease in FATP2. (4) Conclusion: We have identified a new combination enhancing the immune-mediated anticancer therapy in a TNBC mouse model through the reprograming of PMN-MDSCs towards a less immunosuppressive phenotype. These findings prompt the testing of GTN combined with chemotherapies as an adjuvant in TNBC patients experiencing treatment failure.

Canonical and non-canonical functions of NLRP3.

BACKGROUND: Since its discovery, NLRP3 is almost never separated from its major role in the protein complex it forms with ASC, NEK7 and Caspase-1, the inflammasome. This key component of the innate immune response mediates the secretion of proinflammatory cytokines IL-1ß and IL-18 involved in immune response to microbial infection and cellular damage. However, NLRP3 has also other functions that do not involve the inflammasome assembly nor the innate immune response. These non-canonical functions have been poorly studied. Nevertheless, NLRP3 is associated with different kind of diseases probably through its inflammasome dependent function as through its inflammasome independent functions. AIM OF THE REVIEW: The study and understanding of the canonical and non-canonical functions of NLRP3 can help to better understand its involvement in various pathologies. In parallel, the description of the mechanisms of action and regulation of its various functions, can allow the identification of new therapeutic strategies. KEY SCIENTIFIC CONCEPTS OF THE REVIEW: NLRP3 functions have mainly been studied in the context of the inflammasome, in myeloid cells and in totally deficient transgenic mice. However, for several year, the work of different teams has proven that NLRP3 is also expressed in other cell types where it has functions that are independent of the inflammasome. If these studies suggest that NLRP3 could play different roles in the cytoplasm or the nucleus of the cells, the mechanisms underlying NLRP3 non-canonical functions remain unclear. This is why we propose in this review an inventory of the canonical and non-canonical functions of NLRP3 and their impact in different pathologies.

Role of Cytokines and Chemokines in Angiogenesis in a Tumor Context.

During carcinogenesis, tumors set various mechanisms to help support their development. Angiogenesis is a crucial process for cancer development as it drives the creation of blood vessels within the tumor. These newly formed blood vessels insure the supply of oxygen and nutrients to the tumor, helping its growth. The main factors that regulate angiogenesis are the five members of the vascular endothelial growth factor (VEGF) family. Angiogenesis is a hallmark of cancer and has been the target of new therapies this past few years. However, angiogenesis is a complex phenomenon with many redundancy pathways that ensure its maintenance. In this review, we will first describe the consecutive steps forming angiogenesis, as well as its classical regulators. We will then discuss how the cytokines and chemokines present in the tumor microenvironment can induce or block angiogenesis. Finally, we will focus on the therapeutic arsenal targeting angiogenesis in cancer and the challenges they have to overcome.

Hematopoietic Prostaglandin D2 Synthase Controls Tfh/Th2 Communication and Limits Tfh Antitumor Effects.

T follicular helper (Tfh) cells are a subset of CD4+ T cells essential in immunity and have a role in helping B cells produce antibodies against pathogens. However, their role during cancer progression remains unknown. The mechanism of action of Tfh cells remains elusive because contradictory data have been reported on their protumor or antitumor responses in human and murine tumors. Like Tfh cells, Th2 cells are also involved in humoral immunity and are regularly associated with tumor progression and poor prognosis, mainly through their secretion of IL4. Here, we showed that Tfh cells expressed hematopoietic prostaglandin D2 (PGD2) synthase in a pSTAT1/pSTAT3-dependent manner. Tfh cells produced PGD2, which led to recruitment of Th2 cells via the PGD2 receptor chemoattractant receptor homologous molecule expressed on Th type 2 cells (CRTH2) and increased their effector functions. This cross-talk between Tfh and Th2 cells promoted IL4-dependent tumor growth. Correlation between Th2 cells, Tfh cells, and hematopoietic PGD2 synthase was observed in different human cancers and associated with outcome. This study provides evidence that Tfh/Th2 cross-talk through PGD2 limits the antitumor effects of Tfh cells and, therefore, could serve as a therapeutic target.

NLRP6 negatively regulates type 2 immune responses in mice.

BACKGROUND: Inflammasomes are large protein complexes that assemble in the cytosol in response to danger such as tissue damage or infection. Following activation, inflammasomes trigger cell death and the release of biologically active forms of pro-inflammatory cytokines interleukin (IL)-1ß and IL-18. NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammasome is required for IL-18 secretion by intestinal epithelial cells, macrophages, and T cells, contributing to homeostasis and self-defense against pathogenic microbes. However, the involvement of NLRP6 in type 2 lung inflammation remains elusive. METHODS: Wild-type (WT) and Nlrp6-/- mice were used. Birch pollen extract (BPE)-induced allergic lung inflammation, eosinophil recruitment, Th2-related cytokine and chemokine production, airway hyperresponsiveness, and lung histopathology, Th2 cell differentiation, GATA3, and Th2 cytokines expression, were determined. Nippostrongylus brasiliensis (Nb) infection, worm count in intestine, type 2 innate lymphoid cell (ILC2), and Th2 cells in lungs were evaluated. RESULTS: We demonstrate in Nlrp6-/- mice that a mixed Th2/Th17 immune responses prevailed following birch pollen challenge with increased eosinophils, ILC2, Th2, and Th17 cell induction and reduced IL-18 production. Nippostrongylus brasiliensis infected Nlrp6-/- mice featured enhanced early expulsion of the parasite due to enhanced type 2 immune responses compared to WT hosts. In vitro, NLRP6 repressed Th2 polarization, as shown by increased Th2 cytokines and higher expression of the transcription factor GATA3 in the absence of NLRP6. Exogenous IL-18 administration partially reduced the enhanced airways inflammation in Nlrp6-/- mice. CONCLUSIONS: In summary, our data identify NLRP6 as a negative regulator of type 2 immune responses.

Protein Kinase Inhibitor-Mediated Immunoprophylactic and Immunotherapeutic Control of Colon Cancer.

Immunotherapy has allowed major advances in oncology in the past years, in particular with the development of immune checkpoint inhibitors, but the clinical benefits are still limited, particularly in colorectal cancer (CRC). Our scientific approach is based on the search for innovative immunotherapy with a final goal that aims to induce an effective antitumor immune response in CRC. Here, we focused on a multikinase inhibitor, H89. We carried out in vivo experiments based on syngeneic mouse models of colon cancer in BALB/c mice and chemically colon tumorigenesis. Flow cytometry, RNAseq, RT-qPCR, antibody-specific immune cell depletion, and Western blot were used to identify the immune cell type involved in the preventive and antitumor activity of H89. We demonstrated that H89 delays colon oncogenesis and prevents tumor growth. This latter effect seems to involve NK cells. H89 also inhibits colon tumor growth in a T-cell-dependent manner. Analysis of the immune landscape in the tumor microenvironment showed an increase of CD4+ Th1 cells and CD8+ cytotoxic T cells but a decrease of CD4+ Treg cell infiltration. Mechanistically, we showed that H89 could promote naïve CD4+ T-cell differentiation into Th1, a decrease in Treg differentiation, and an increase in CD8+ T-cell activation and cytotoxicity ex vivo. Furthermore, H89 induced overexpression of genes involved in antitumor immune response, such as IL-15RA, which depletion counteracts the antitumor effect of H89. We also found that H89 regulated Akt/PP2A pathway axis, involved in TCR and IL-15 signaling transduction. Our findings identify the H89 as a potential strategy for immune system activation leading to the prevention and treatment of CRC.

Alternative Splicing in Cancer and Immune Cells.

Splicing is a phenomenon enabling the excision of introns from pre-mRNA to give rise to mature mRNA. All the 20,000 genes of the human genome are concerned by this mechanism. Nevertheless, it is estimated that the proteome is composed of more than 100,000 proteins. How to go from 20,000 genes to more than 100,000 proteins? Alternative splicing (AS) is in charge of this diversity of proteins. AS which is found in most of the cells of an organism, participates in normal cells and in particular in immune cells, in the regulation of cellular behavior. In cancer, AS is highly dysregulated and involved in almost all of the hallmarks that characterize tumor cells. In view of the close link that exists between tumors and the immune system, we present in this review the literature relating to alternative splicing and immunotherapy. We also provide a global but not exhaustive view of AS in the immune system and tumor cells linked to the events that can lead to AS dysregulation in tumors.

Impact of Lipid Metabolism on Antitumor Immune Response.

Over the past decade, metabolic reprogramming has been defined as a hallmark of cancer. More recently, a large number of studies have demonstrated that metabolic reprogramming can modulate the differentiation and functions of immune cells, and thus modify the antitumor response. Increasing evidence suggests that modified energy metabolism could be responsible for the failure of antitumor immunity. Indeed, tumor-infiltrating immune cells play a key role in cancer, and metabolic switching in these cells has been shown to help determine their phenotype: tumor suppressive or immune suppressive. Recent studies in the field of immunometabolism focus on metabolic reprogramming in the tumor microenvironment (TME) by targeting innate and adaptive immune cells and their associated anti- or protumor phenotypes. In this review, we discuss the lipid metabolism of immune cells in the TME as well as the effects of lipids; finally, we expose the link between therapies and lipid metabolism.

Recruitment and activation of type 3 innate lymphoid cells promote antitumor immune responses.

Tumors poorly infiltrated by T cells are more resistant to immunogenic chemotherapies and checkpoint inhibition than highly infiltrated tumors. Using murine models, we found that CCR6+ type 3 innate lymphoid cells (ILC3s) can trigger an increase in the number of T cells infiltrating a tumor. Shortly after administration of cisplatin chemotherapy, production of the chemokine CCL20 and proinflammatory cytokine IL-1ß at the tumor site led to the recruitment and activation of ILC3s. Within the tumor, ILC3 production of the chemokine CXCL10 was responsible for the recruitment of CD4+ and CD8+ T lymphocytes to the tumor. ILC3-dependent infiltration of T cells was essential for antitumor immune responses and increased the efficacy of checkpoint inhibition. Thus, we reveal an essential role of CCL20 and IL-1ß, which promote ILC3-dependent antitumor immunity and enhance tumor sensitivity to immunotherapy.

CD4 T cell-intrinsic STING signaling controls the differentiation and effector functions of TH1 and TH9 cells.

BACKGROUND: While stimulator of interferon genes (STING) activation in innate immune cells of the tumor microenvironment can result in CD8 T cell-dependent antitumor immunity, whether STING signaling affects CD4 T-cell responses remains elusive. METHODS: Here, we tested whether STING activation modulated the effector functions of CD4 T cells in vivo by analyzing tumor-infiltrating CD4 T cells and evaluating the contribution of the CD4 T cell-derived cytokines in the antitumor activity of the STING ligand 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) in two mouse tumor models. We performed ex vivo experiments to assess the impact of STING activation on CD4 T-cell differentiation and investigate the underlying molecular mechanisms. Finally, we tested whether STING activation enhances TH9 cell antitumor activity against mouse melanoma upon adoptive transfer. RESULTS: We found that activation of STING signaling cell-intrinsically enhances the differentiation and antitumor functions of TH1 and TH9 cells by increasing their respective production of interferon gamma (IFN-γ) and interleukin-9. IRF3 and type I interferon receptors (IFNARs) are required for the STING-driven enhancement of TH1 cell differentiation. However, STING activation favors TH9 cell differentiation independently of the IFNARs/IRF3 pathway but through mammalian target of rapamycin (mTOR) signaling, underscoring that STING activation differentially affects the fate of distinct CD4 T-cell subsets. The therapeutic effect of STING activation relies on TH1 and TH9-derived cytokines, and STING activation enhances the antitumor activity of TH9 cells upon adoptive transfer. CONCLUSION: Our results reveal the STING signaling pathway as a therapeutic target to boost CD4 T-cell effector functions and antitumor immunity.

Follicular helper-T cells restore CD8+-dependent antitumor immunity and anti-PD-L1/PD-1 efficacy.

BACKGROUND: T follicular helper cells (Tfh) are essential to shape B cell response during germinal center formation. Tfh accumulation has been reported in various human cancers, with positive or negative prognostic roles. However, the mechanisms explaining the accumulation of Tfh and their role in cancer remain obscure. METHODS: In vitro differentiated and mouse cell sorted Tfh phenotype was evaluated by flow cytometry and quantitative PCR (qPCR). Antitumor effect of Tfh was evaluated by adoptive transfer in different tumor-bearing mice models. The involvement of immune cells, cytokines and chemokines was evaluated, using depleting antibodies. Chemokines and cytokines expression and production were evaluated by qPCR and ELISA. In human, the impact of immune cells and chemokines on survival was evaluated by analyzing transcriptomic data from public databases and from our own patient cohorts. RESULTS: In this study, we show that Tfh exert an antitumor immune effect in a CD8+-dependent manner. Tfh produce interleukin-21, which sustains proliferation, viability, cytokine production and cytotoxic functions of exhausted T cells. The presence of Tfh is required for efficacy of antiprogrammed cell death ligand-1 therapy. Tfh accumulate in the tumor bed and draining lymph nodes in different mouse cancer models. This recruitment is due to the capacity of transforming growth factor ß to drive Chemokine (C-X-C motif) Ligand 13 expression, a chemoattractant of Tfh, by intratumor CD8+ T cells. Accumulation of Tfh and exhausted CD8+ T cells predicts cancer outcome in various cancer types. In patients treated with anti-programmed cell death-1 mAb, accumulation of Tfh and CD8+ at the tumor site is associated with outcome. CONCLUSION: This study provides evidence that CD8+/Tfh crosstalk is important in shaping antitumor immune response generated by immunotherapy.

Modulation of CD4 T Cell Response According to Tumor Cytokine Microenvironment.

The advancement of knowledge on tumor biology over the past decades has demonstrated a close link between tumor cells and cells of the immune system. In this context, cytokines have a major role because they act as intermediaries in the communication into the tumor bed. Cytokines play an important role in the homeostasis of innate and adaptive immunity. In particular, they participate in the differentiation of CD4 T lymphocytes. These cells play essential functions in the anti-tumor immune response but can also be corrupted by tumors. The differentiation of naïve CD4 T cells depends on the cytokine environment in which they are activated. Additionally, at the tumor site, their activity can also be modulated according to the cytokines of the tumor microenvironment. Thus, polarized CD4 T lymphocytes can see their phenotype evolve, demonstrating functional plasticity. Knowledge of the impact of these cytokines on the functions of CD4 T cells is currently a source of innovation, for therapeutic purposes. In this review, we discuss the impact of the major cytokines present in tumors on CD4 T cells. In addition, we summarize the main therapeutic strategies that can modulate the CD4 response through their impact on cytokine production.

The Tumor Microenvironment Impairs Th1 IFNγ Secretion through Alternative Splicing Modifications of Irf1 Pre-mRNA.

It is clearly established that the immune system can affect cancer response to therapy. However, the influence of the tumor microenvironment (TME) on immune cells is not completely understood. In this respect, alternative splicing is increasingly described to affect the immune system. Here, we showed that the TME, via a TGFß-dependent mechanism, increased alternative splicing events and induced the expression of an alternative isoform of the IRF1 transcription factor (IRF1Δ7) in Th1 cells. We found that the SFPQ splicing factor (splicing factor, proline- and glutamine-rich) was responsible for the IRF1Δ7 production. We also showed, in both mice and humans, that the IRF1 alternative isoform altered the full-length IRF1 transcriptional activity on the Il12rb1 promoter, resulting in decreased IFNγ secretion in Th1 cells. Thus, the IRF1Δ7 isoform was increased in the TME, and inhibiting IRF1Δ7 expression could potentiate Th1 antitumor responses.

Cleaved Caspase-3 Transcriptionally Regulates Angiogenesis-Promoting Chemotherapy Resistance.

Caspases are well known for their role in apoptosis. Recently, nonapoptotic roles of caspases have been identified, however, these noncanonical roles are not well documented and the mechanisms involved are not fully understood. Here, we studied the role of cleaved caspase-3 using human- and mouse-proficient caspase-3 cancer cell lines and human-deficient caspase-3 cancer cells. Cleaved caspase-3 functioned as a transcription factor and directly bound to DNA. A DNA-binding domain was identified in the small subunit of caspase-3 and an active conformation was essential for caspase-3 transcriptional activity. Caspase-3 DNA binding enhanced angiogenesis by upregulating the expression of proangiogenic genes and by activating pathways that promoted endothelial cell activation. Some proapoptotic genes were downregulated in caspase-3-proficient cells. Inhibiting caspase-3 increased the efficacy of chemotherapy and decreased spontaneous tumor development. These data highlight a novel nonapoptotic role of caspase-3 and suggest that cleaved caspase-3 could be a new therapeutic target in cancer. SIGNIFICANCE: These findings report a noncanonical function of caspase-3 by demonstrating its ability to transcriptionally regulate the VEGFR pathway.

Transcriptional Programs Underlying Cd4 T Cell Differentiation and Functions.

Understanding the basis of cellular differentiation is a fundamental issue in developmental biology but also for the comprehension of pathological processes. In fact, the palette of developmental decisions for naive CD4 T cells is a critical aspect of the development of appropriate immune responses which could control infectious processes or cancer growth. However, the current accumulation of data on CD4 T cells biology reveals a complex world with different helper populations. Naive CD4 T cells can differentiate into different subtypes in response to cytokine stimulation. This stimulation involves a complex transcriptional network implicating the activation of Signal Transducer and Activator of Transcription but also master regulator transcription factors allowing the functions of each helper T lymphocyte subtype. In this review, we will present an overview of the transcriptional regulation which controls process of helper T cells differentiation. We will focus on the role of initiator transcriptional factors and on master regulators but also on other nonspecific transcriptional factors which refine the T helper polarization to stabilize or modulate the differentiation program.