Targeting MICA/B with cytotoxic therapeutic antibodies leads to tumor control.

Background: MICA and MICB are tightly regulated stress-induced proteins that trigger the immune system by binding to the activating receptor NKG2D on cytotoxic lymphocytes. MICA and MICB are highly polymorphic molecules with prevalent expression on several types of solid tumors and limited expression in normal/healthy tissues, making them attractive targets for therapeutic intervention. Methods: We have generated a series of anti-MICA and MICB cross-reactive antibodies with the unique feature of binding to the most prevalent isoforms of both these molecules. Results: The anti-MICA and MICB antibody MICAB1, a human IgG1 Fc-engineered monoclonal antibody (mAb), displayed potent antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of MICA/B-expressing tumor cells in vitro. However, it showed insufficient efficiency against solid tumors in vivo, which prompted the development of antibody-drug conjugates (ADC). Indeed, optimal tumor control was achieved with MICAB1-ADC format in several solid tumor models, including patient-derived xenografts (PDX) and carcinogen-induced tumors in immunocompetent MICAgen transgenic mice. Conclusions: These data indicate that MICA and MICB are promising targets for cytotoxic immunotherapy.

NKp46 is a diagnostic biomarker and may be a therapeutic target in gastrointestinal T-cell lymphoproliferative diseases: a CELAC study.

OBJECTIVES: Primary GI T-cell lymphoproliferative diseases (T-LPD) are heterogeneous entities, which raise difficult diagnosis and therapeutic challenges. We have recently provided evidences that lymphomas complicating coeliac disease (CD) arise from innate-like lymphocytes, which may carry NK receptors (NKRs). DESIGN: NKRs expression was compared by flow cytometry in intraepithelial lymphocytes (IEL) from CD, type I or type II refractory CD (RCD). NKp46 was next assessed by immunohistochemistry in paraffin-embedded biopsies from 204 patients with CD, RCDI, RCDII or GI T-cell lymphomas and from a validation cohort of 61 patients. The cytotoxic properties of an anti-NKp46 monoclonal antibody conjugated to pyrrolobenzodiazepine (PBD) was tested ex vivo in human primary tumour cells isolated from fresh duodenal biopsies. RESULTS: NKp46 (but not CD94, NKG2A, NKG2C, NKG2D) was significantly more expressed by malignant RCDII IEL than by normal IEL in CD and RCDI. In paraffin biopsies, detection of >25 NKp46+ IEL per 100 epithelial cells discriminated RCDII from CD and RCDI. NKp46 was also detected in enteropathy-associated T-cell lymphomas (EATL, 24/29) and in monomorphic epitheliotropic intestinal T-cell lymphomas (MEITL, 4/4) but not in indolent T-LPD (0/15). Treatment with anti-NKp46-PBD could efficiently and selectively kill human NKp46+ primary IEL ex vivo. CONCLUSION: NKp46 is a novel biomarker useful for diagnosis and therapeutic stratification of GI T-LPD. Strong preclinical rationale identifies anti-NKp46-PBD as a promising therapy for RCDII, EATL and MEITL.

TP53INP1 deficiency maintains murine B lymphopoiesis in aged bone marrow through redox-controlled IL-7R/STAT5 signaling.

Bone marrow (BM) produces all blood and immune cells deriving from hematopoietic stem cells (HSCs). The decrease of immune cell production during aging is one of the features of immunosenescence. The impact of redox dysregulation in BM aging is still poorly understood. Here we use TP53INP1-deficient (KO) mice endowed with chronic oxidative stress to assess the influence of aging-associated redox alterations in BM homeostasis. We show that TP53INP1 deletion has no impact on aging-related accumulation of HSCs. In contrast, the aging-related contraction of the lymphoid compartment is mitigated in TP53INP1 KO mice. B cells that accumulate in old KO BM are differentiating cells that can mature into functional B cells. Importantly, this phenotype results from B cell-intrinsic events associated with defective redox control. Finally, we show that oxidative stress in aged TP53INP1-deficient mice maintains STAT5 expression and activation in early B cells, driving high Pax5 expression, which provides a molecular mechanism for maintenance of B cell development upon aging.

JAM-C Identifies Src Family Kinase-Activated Leukemia-Initiating Cells and Predicts Poor Prognosis in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) originates from hematopoietic stem and progenitor cells that acquire somatic mutations, leading to disease and clonogenic evolution. AML is characterized by accumulation of immature myeloid cells in the bone marrow and phenotypic cellular heterogeneity reflective of normal hematopoietic differentiation. Here, we show that JAM-C expression defines a subset of leukemic cells endowed with leukemia-initiating cell activity (LIC). Stratification of de novo AML patients at diagnosis based on JAM-C-expressing cells frequencies in the blood served as an independent prognostic marker for disease outcome. Using publicly available leukemic stem cell (LSC) gene expression profiles and gene expression data generated from JAM-C-expressing leukemic cells, we defined a single cell core gene expression signature correlated to JAM-C expression that reveals LSC heterogeneity. Finally, we demonstrated that JAM-C controls Src family kinase (SFK) activation in LSC and that LIC with exacerbated SFK activation was uniquely found within the JAM-C-expressing LSC compartment. Cancer Res; 77(23); 6627-40. ©2017 AACR.

BTN3A molecules considerably improve Vγ9Vδ2T cells-based immunotherapy in acute myeloid leukemia.

Given their recognized ability to kill acute myeloid leukemia (AML) blasts both in vitro and in vivo, Vγ9Vδ2 T cells are of growing interest in the design of new strategies of immunotherapy. We show that the Butyrophilin3A (BTN3A, CD277) subfamily is a critical determinant of Vγ9Vδ2 TCR-mediated recognition of human primary AML blasts ex vivo. Moreover, anti-BTN3A 20.1 agonist monoclonal antibodies (mAbs) can trigger BTN3A on AML blasts leading to further enhanced Vγ9Vδ2 T cell-mediated killing, but this mAb had no enhancing effect upon NK cell-mediated killing. We show that monocytic differentiation of primary AML blasts accounts for their AminoBisphosphonate (N-BP)-mediated sensitization to Vγ9Vδ2 T cells. In addition, anti-BTN3A 20.1 mAbs could specifically sensitize resistant blasts to Vγ9Vδ2 T cells lysis and overcome the poor effect of N-BP treatment on those blasts. We confirmed the enhancement of Vγ9Vδ2 T cells activity by anti-BTN3A 20.1 mAb using a human AML xenotransplantation mouse model. We showed that anti-BTN3A 20.1 mAb combined with Vγ9Vδ2 T cells immunotherapy could increase animal survival and decrease the leukemic burden in blood and bone marrow. These findings could be of great interest in the design of new immunotherapeutic strategies for treating AML.

Protective mitochondrial transfer from bone marrow stromal cells to acute myeloid leukemic cells during chemotherapy.

Here we demonstrate that in a niche-like coculture system, cells from both primary and cultured acute myeloid leukemia (AML) sources take up functional mitochondria from murine or human bone marrow stromal cells. Using different molecular and imaging approaches, we show that AML cells can increase their mitochondrial mass up to 14%. After coculture, recipient AML cells showed a 1.5-fold increase in mitochondrial adenosine triphosphate production and were less prone to mitochondrial depolarization after chemotherapy, displaying a higher survival. This unidirectional transfer enhanced by some chemotherapeutic agents required cell-cell contacts and proceeded through an endocytic pathway. Transfer was greater in AML blasts compared with normal cord blood CD34(+) cells. Finally, we demonstrate that mitochondrial transfer was observed in vivo in an NSG immunodeficient mouse xenograft model and also occurred in human leukemia initiating cells and progenitors. As mitochondrial transfer provides a clear survival advantage following chemotherapy and a higher leukemic long-term culture initiating cell potential, targeting mitochondrial transfer could represent a future therapeutic target for AML treatment.

Defects in mitophagy promote redox-driven metabolic syndrome in the absence of TP53INP1.

The metabolic syndrome covers metabolic abnormalities including obesity and type 2 diabetes (T2D). T2D is characterized by insulin resistance resulting from both environmental and genetic factors. A genome-wide association study (GWAS) published in 2010 identified TP53INP1 as a new T2D susceptibility locus, but a pathological mechanism was not identified. In this work, we show that mice lacking TP53INP1 are prone to redox-driven obesity and insulin resistance. Furthermore, we demonstrate that the reactive oxygen species increase in TP53INP1-deficient cells results from accumulation of defective mitochondria associated with impaired PINK/PARKIN mitophagy. This chronic oxidative stress also favors accumulation of lipid droplets. Taken together, our data provide evidence that the GWAS-identified TP53INP1 gene prevents metabolic syndrome, through a mechanism involving prevention of oxidative stress by mitochondrial homeostasis regulation. In conclusion, this study highlights TP53INP1 as a molecular regulator of redox-driven metabolic syndrome and provides a new preclinical mouse model for metabolic syndrome clinical research.

The thymus-specific serine protease TSSP/PRSS16 is crucial for the antitumoral role of CD4(+) T cells.

In cancer, immune cells can play conflicting roles, either protective, by elimination of tumor cells during immune surveillance, or detrimental, by promoting carcinogenesis during inflammation. We report here that the thymus-specific serine protease (TSSP), which is involved in CD4(+) T cell maturation in the thymus, exerts a tumor suppressor activity. Mice genetically deficient for TSSP are highly prone to spontaneous cancer development. The absence of TSSP also increases the rate of induced colitis-associated colorectal (CAC) tumor formation, through exacerbated colon inflammation. Adoptive transfer of T cells in various combinations (CD4(+) and CD8(+) from wild-type and/or knockout mice) into T cell-deficient mice showed that the TSSP-deficient CD4(+) T cell compartment promotes tumor development, associated with high levels of the cytokine IL-17A. Inhibition of IL-17A during CAC tumor formation prevents the increased carcinogenesis and colic immune disequilibrium observed in TSSP-deficient mice. Therefore, our data demonstrate that antitumoral immune surveillance requires thymic TSSP-driven production of CD4(+) T cells contributing to inflammatory balance.

Therapeutic targeting of c-Myc in T-cell acute lymphoblastic leukemia, T-ALL.

T-ALL patients treated with intensive chemotherapy achieve high rates of remission. However, frequent long-term toxicities and relapses into chemotherapy-refractory tumors constitute major clinical challenges which could be met by targeted therapies. c-MYC is a central oncogene in T-ALL, prompting the exploration of the efficacy of MYC inhibitors such as JQ1 (BET-bromodomain inhibitor), and SAHA (HDAC inhibitor). Using a standardized ex vivo drug screening assay, we show here that JQ1 and SAHA show competitive efficiency compared to inhibitors of proteasome, PI3K/AKT/mTOR and NOTCH pathways, and synergize in combination with Vincristine. We also compared for the first time the in vivo relevance of such associations in mice xenografted with human primary T-ALLs. Our data indicate that although treatments combining JQ1 or SAHA with chemotherapeutic regimens might represent promising developments in T-ALL, combinations will need to be tailored to specific subgroups of responsive patients, the profiles of which still remain to be precisely defined.

Primary B-CLL resistance to NK cell cytotoxicity can be overcome in vitro and in vivo by priming NK cells and monoclonal antibody therapy.

Despite recent advances with monoclonal antibody therapy, chronic lymphocytic leukemia (CLL) remains incurable. Natural killer (NK) cells are potent antitumoral effectors, particularly against hematological malignancies. Defective recognition of B-CLL leukemic cells by NK cells has been previously described. Here, we deciphered the mechanisms that hamper NK cell-mediated clearance of B-CLL and evaluated the potential of NK cells as therapeutic tools for treatment of CLL. First of all, leukemic B cells resemble to normal B cells with a weak expression of ligands for NK receptors. Conversely, NK cells from B-CLL patients were functionally and phenotypically competent, despite a decrease of expression of the activating receptor NKp30. Consequently, resting allogeneic NK cells were unable to kill leukemic B cells in vitro. These data suggest that patients' NK cells cannot initiate a proper immune reaction due to a lack of leukemic cell recognition. We next set up a xenotransplantation mouse model to study NK-CLL cell interactions. Together with our in vitro studies, in vivo data revealed that activation of NK cells is required in order to control B-CLL and that activated NK cells synergize to enhance rituximab effect on tumor load. This study points out the requirements for immune system manipulation for treatment of B-CLL in combination with monoclonal antibody therapy.

Saccharomyces boulardii improves intestinal cell restitution through activation of the α2ß1 integrin collagen receptor.

Intestinal epithelial cell damage is frequently seen in the mucosal lesions of inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the cessation of inflammation and the migration of enterocytes to repair the damaged epithelium. Lyophilized Saccharomyces boulardii (Sb, Biocodex) is a nonpathogenic yeast widely used as a therapeutic agent for the treatment and prevention of diarrhea and other gastrointestinal disorders. In this study, we determined whether Sb could accelerate enterocyte migration. Cell migration was determined in Sb force-fed C57BL6J mice and in an in vitro wound model. The impact on α2ß1 integrin activity was assessed using adhesion assays and the analysis of α2ß1 mediated signaling pathways both in vitro and in vivo. We demonstrated that Sb secretes compounds that enhance the migration of enterocytes independently of cell proliferation. This enhanced migration was associated with the ability of Sb to favor cell-extracellular matrix interaction. Indeed, the yeast activates α2ß1 integrin collagen receptors. This leads to an increase in tyrosine phosphorylation of cytoplasmic molecules, including focal adhesion kinase and paxillin, involved in the integrin signaling pathway. These changes are associated with the reorganization of focal adhesion structures. In conclusion Sb secretes motogenic factors that enhance cell restitution through the dynamic regulation of α2ß1 integrin activity. This could be of major importance in the development of novel therapies targeting diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.

Absence of tumor suppressor tumor protein 53-induced nuclear protein 1 (TP53INP1) sensitizes mouse thymocytes and embryonic fibroblasts to redox-driven apoptosis.

The p53-transcriptional target TP53INP1 is a potent stress-response protein promoting p53 activity. We previously showed that ectopic overexpression of TP53INP1 facilitates cell cycle arrest as well as cell death. Here we report a study investigating cell death in mice deficient for TP53INP1. Surprisingly, we found enhanced stress-induced apoptosis in TP53INP1-deficient cells. This observation is underpinned in different cell types in vivo (thymocytes) and in vitro (thymocytes and MEFs), following different types of injury inducing either p53-dependent or -independent cell death. Nevertheless, absence of TP53INP1 is unable to overcome impaired cell death of p53-deficient thymocytes. Stress-induced ROS production is enhanced in the absence of TP53INP1, and antioxidant NAC complementation abolishes increased sensitivity to apoptosis of TP53INP1-deficient cells. Furthermore, antioxidant defenses are defective in TP53INP1-deficient mice in correlation with ROS dysregulation. Finally, we show that autophagy is reduced in TP53INP1-deficient cells both at the basal level and upon stress. Altogether, these data show that impaired ROS regulation in TP53INP1-deficient cells is responsible for their sensitivity to induced apoptosis. In addition, they suggest that this sensitivity could rely on a defect of autophagy. Therefore, these data emphasize the role of TP53INP1 in protection against cell injury.

Mutant mouse models of oxidative stress.

Oxidative stress corresponds to an excess in reactive oxygen species (ROS) including free radicals which are highly reactive with cellular constituents. Thereby ROS induce damage to DNA, proteins and lipids, which are all involved in the etiology of numerous pathologies such as cancer. To prevent potential damage, a tight regulation of ROS level is achieved through numerous enzyme systems and small molecules such as glutathione and vitamin C. Mutant mouse models targeting antioxidant enzymes have confirmed their essential role in ROS level control, and have shown a limited redundancy of their activity. Additionally, a number of other mutant mouse models exhibit increased ROS levels, suggesting an antioxidant role for the corresponding targeted gene. This is the case for mice deficient for the transcription factors p53, JunD, FoxOs, and HIF-2alpha, which are involved in the modulation of antioxidant enzymes expression. Mice deficient either for the stress factor TP53INP1, which is a target of p53, or for ATM involved in DNA damage sensoring, also show a constitutive oxidative stress. Finally, the last reported case of mice with a permanent oxidative stress targets Bmi which is a transcriptional repressor of the polycomb family. Interestingly, most of these "oxidative stressed mice" either spontaneously develop cancers or are more susceptible than wild-type to tumor-induced protocols. Altogether, these models markedly reinforce the causal link between oxidative stress and cancer. In the future, they will be helpful tools for basic research aimed at unraveling the interplay between redox control actors as well as their relative importance. In addition, these oxidative stressed mouse models may be useful for applied research in particular in preclinical assays where redox status regulation is absolutely required.

Epithelial vanin-1 controls inflammation-driven carcinogenesis in the colitis-associated colon cancer model.

BACKGROUND: Vanin-1 is an epithelial pantetheinase that provides cysteamine to tissue and regulates response to stress. Vanin-1 is expressed by enterocytes, and its absence limits intestinal epithelial cell production of proinflammatory signals. A link between chronic active inflammation and cancer is illustrated in patients with ulcerative colitis, who have an augmented risk of developing colorectal cancer. Indeed, sustained inflammation provides advantageous growth conditions to tumors. We examined whether epithelial cells affect tumorigenesis through vanin-1-dependent modulation of colonic inflammation. METHODS: To vanin-1(-/-) mice, we applied the colitis-associated cancer (CAC) protocol, which combines injection of azoxymethane (AOM) with repeated administrations of dextran sodium sulfate (DSS). We numbered tumors and quantified macrophage infiltration and molecular markers of cell death and proliferation. We also tested DSS-induced colitis. We scored survival, tissue damages, proinflammatory cytokine production, and tissue regeneration. Finally, we explored activation pathways by biochemical analysis on purified colonic epithelial cells (CECs) and in situ immunofluorescence. RESULTS: Vanin-1(-/-) mice displayed a drastically reduced incidence of colorectal cancer in the CAC protocol and manifested mild clinical signs of DSS-induced colitis. The early impact of vanin-1 deficiency on tumor induction was directly correlated to the amount of inflammation and subsequent epithelial proliferation rather than cell death rate; all this was linked to the modulation of NF-kappaB pathway activation in CECs. CONCLUSIONS: These results emphasize the importance of the intestinal epithelium in the control of mucosal inflammation acting as a cofactor in carcinogenesis. This might lead to novel anti-inflammatory strategies useful in cancer therapy.

Distinct thresholds govern Myc's biological output in vivo.

Deregulated Myc triggers a variety of intrinsic tumor suppressor programs that serve to restrain Myc's oncogenic potential. Since Myc activity is also required for normal cell proliferation, activation of intrinsic tumor suppression must be triggered only when Myc signaling is oncogenic. However, how cells discriminate between normal and oncogenic Myc is unknown. Here we show that distinct threshold levels of Myc govern its output in vivo: low levels of deregulated Myc are competent to drive ectopic proliferation of somatic cells and oncogenesis, but activation of the apoptotic and ARF/p53 intrinsic tumor surveillance pathways requires Myc overexpression. The requirement to keep activated oncogenes at a low level to avoid engaging tumor suppression is likely an important selective pressure governing the early stages of tumor microevolution.

Vanin-1 licenses inflammatory mediator production by gut epithelial cells and controls colitis by antagonizing peroxisome proliferator-activated receptor gamma activity.

Colitis involves immune cell-mediated tissue injuries, but the contribution of epithelial cells remains largely unclear. Vanin-1 is an epithelial ectoenzyme with a pantetheinase activity that provides cysteamine/cystamine to tissue. Using the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-colitis model we show here that Vanin-1 deficiency protects from colitis. This protection is reversible by administration of cystamine or bisphenol A diglycidyl ether, a peroxisome proliferator-activated receptor (PPAR)gamma antagonist. We further demonstrate that Vanin-1, by antagonizing PPARgamma, licenses the production of inflammatory mediators by intestinal epithelial cells. We propose that Vanin-1 is an epithelial sensor of stress that exerts a dominant control over innate immune responses in tissue. Thus, the Vanin-1/pantetheinase activity might be a new target for therapeutic intervention in inflammatory bowel disease.

LAT regulates gammadelta T cell homeostasis and differentiation.

LAT (linker for activation of T cells) is essential for T cell receptor signaling. Mice homozygous for a mutation of the three C-terminal LAT tyrosine residues showed a block in alphabeta T cell development and a partially impaired gammadelta T cell development. Without intentional immunization, they accumulated gammadelta T cells in the spleen and lymph nodes that chronically produced T helper type 2 cytokines in large amounts, and caused the maturation of plasma cells secreting immunoglobulin E (IgE) and IgG1. These effects are very similar to that triggered in the alphabeta lineage by a mutation involving a distinct LAT tyrosine. Thus, LAT is an essential regulator of T cell homeostasis and terminal differentiation.