Nathalie Bonnefoy

Protein kinase Cδ deficiency causes mendelian systemic lupus erythematosus with B cell-defective apoptosis and hyperproliferation

OBJECTIVE Systemic lupus erythematosus (SLE) is a prototype autoimmune disease that is assumed to occur via a complex interplay of environmental and genetic factors. Rare causes of monogenic SLE have been described, providing unique insights into fundamental mechanisms of immune tolerance. The aim of this study was to identify the cause of an autosomal-recessive form of SLE. METHODS We studied 3 siblings with juvenile-onset SLE from 1 consanguineous kindred and used next-generation sequencing to identify mutations in the disease-associated gene. We performed extensive biochemical, immunologic, and functional assays to assess the impact of the identified mutations on B cell biology. RESULTS We identified a homozygous missense mutation in PRKCD, encoding protein kinase δ (PKCδ), in all 3 affected siblings. Mutation of PRKCD resulted in reduced expression and activity of the encoded protein PKCδ (involved in the deletion of autoreactive B cells), leading to resistance to B cell receptor- and calcium-dependent apoptosis and increased B cell proliferation. Thus, as for mice deficient in PKCδ, which exhibit an SLE phenotype and B cell expansion, we observed an increased number of immature B cells in the affected family members and a developmental shift toward naive B cells with an immature phenotype. CONCLUSION Our findings indicate that PKCδ is crucial in regulating B cell tolerance and preventing self-reactivity in humans, and that PKCδ deficiency represents a novel genetic defect of apoptosis leading to SLE.

IL-17A is produced by breast cancer TILs and promotes chemoresistance and proliferation through ERK1/2.

The proinflammatory cytokine Interleukin 17A (hereafter named IL–17A) or IL-17A producing cells are elevated in breast tumors environment and correlate with poor prognosis. Increased IL-17A is associated with ER(−) or triple negative tumors and reduced Disease Free Survival. However, the pathophysiological role of IL-17A in breast cancer remains unclear although several studies suggested its involvement in cancer cell dissemination. Here we demonstrated that a subset of breast tumors is infiltrated with IL-17A-producing cells. Increased IL-17A seems mainly associated to ER(−) and triple negative/basal-like tumors. Isolation of tumor infiltrating T lymphocytes (TILs) from breast cancer biopsies revealed that these cells secreted significant amounts of IL-17A. We further established that recombinant IL-17A recruits the MAPK pathway by upregulating phosphorylated ERK1/2 in human breast cancer cell lines thereby promoting proliferation and resistance to conventional chemotherapeutic agents such as docetaxel. We also confirmed here that recombinant IL-17A stimulates migration and invasion of breast cancer cells as previously reported. Importantly, TILs also induced tumor cell proliferation, chemoresistance and migration and treatment with IL-17A-neutralizing antibodies abrogated these effects. Altogether these results demonstrated the pathophysiological role of IL-17A-producing cell infiltrate in a subset of breast cancers. Therefore, IL-17A appears as potential therapeutic target for breast cancer.

IL-17A and its homologs IL-25/IL-17E recruit the c-RAF/S6 kinase pathway and the generation of pro-oncogenic LMW-E in breast cancer cells

Pro-inflammatory IL-17 cytokines were initially described for their pathogenic role in chronic inflammatory diseases and subsequent accumulating evidence indicated their involvement in carcinogenesis. In the present study we report that IL-17A and IL-17E receptors subunits mRNA expressions are upregulated in breast cancers versus normal samples. IL-17E, which is undetectable in most normal breast tissues tested, seems more expressed in some tumors. Investigation of the molecular signaling following stimulation of human breast cancer cell lines with IL-17A and IL-17E showed that both cytokines induced the phosphorylation of c-RAF, ERK1/2 and p70 S6 Kinase were involved in the proliferation and survival of tumor cells. Accordingly, IL-17A and IL-17E promoted resistance to Docetaxel and failed to induce apoptosis as previously reported for IL-17E. Interestingly, we also revealed that both cytokines induced the generation of tumorogenic low molecular weight forms of cyclin E (LMW-E), which high levels correlated strongly with a poor survival in breast cancer patients. These results show for the first time some of the molecular pathways activated by IL-17A and IL-17E that may participate to their pro-oncogenic activity in breast cancers.

The IL-17B-IL-17 receptor B pathway promotes resistance to paclitaxel in breast tumors through activation of the ERK1/2 pathway

Interleukin 17B (IL-17B) is a pro-inflammatory cytokine that belongs to the IL-17 cytokines family and binds to IL-17 receptor B (IL-17RB). Here we found that high expression of IL-17B and IL-17RB is associated with poor prognosis in patients with breast cancer and that IL-17B expression upregulation is specifically associated with poorer survival in patients with basal-like breast cancer. We thus focused on IL-17B role in breast cancer by using luminal and triple negative (TN)/basal-like tumor cell lines. We found that IL-17B induces resistance to conventional chemotherapeutic agents. In vivo, IL-17B induced resistance to paclitaxel and treatment with an anti-IL-17RB neutralizing antibody completely restored breast tumor chemosensitivity, leading to tumor shrinkage. We next focused on the signaling pathways activated in human breast cancer cell lines upon incubation with IL-17B. We observed that IL-17B induces ERK1/2 pathway activation, leading to upregulation of anti-apoptotic proteins of the BCL-2 family. IL-17B-induced chemoresistance was completely abolished by incubation with PD98059, an inhibitor of the MAPK/ERK pathway, indicating that the ERK pathway plays a crucial role. Altogether our results emphasize the role of the IL-17B/IL-17RB signaling pathway in breast tumors and identify IL-17B and its receptor as attractive therapeutic targets for potentiating breast cancer chemotherapy.

Abstract 1602: Generation of anti-IL-17B antibodies neutralizing IL-17B-mediated alterations of the immune microenvironment, promotion of tumor cell initiating capacity and chemoresistance

Interleukin 17B (IL-17B) is a pro-inflammatory cytokine that belongs to a family encompassing 6 interleukins (IL-17A to F) and binds to the IL-17 receptor B (IL-17RB). Recently, amplified IL-17B/IL-17RB signaling was found critical for breast and pancreatic tumorigenesis and elevated expression of IL-17RB has been associated with the shortest survival rates in patients with breast or pancreatic cancer. Using IL-17B knock-out (IL-17B KO) mice we demonstrate here that melanoma, fibrosarcoma and breast cancer cell tumorigenicity is strongly impaired in immunocompetent IL-17B KO mice compared to WT littermates, including a large number of tumor free mice. Reduced tumor incidence in IL-17B KO mice is associated with alterations of the immune tumor microenvironment especially within innate lymphocyte and myeloid sub-populations. We further demonstrate that IL-17B is a key cytokine shaping the tumor initiating cancer cell niche. Indeed, MDA-MB-468 human breast cancer cells overexpressing IL-17B exhibit 10 times higher frequency of tumor initiating cells when xenografted at a serial limiting dilution in nude mice. Tumor progression is, again, associated with alterations of NK cells within the tumor microenvironment and with increased percentages of CD44hi/CD24lo tumor cells, a phenotype associated with breast cancer stem cells (CSC). This is associated with resistance to conventional chemotherapeutic agents such as taxol, an effect that is totally abrogated by disrupting IL-17B-IL-17RB signaling with a neutralizing antibody. Altogether our results point out the key role of IL-17B in regulating the immune microenvironment as well as cardinal features of CSC, one of the alleged causes of resistance to therapy and tumor relapse. Thereby, IL-17B and its receptor appear as potential therapeutic targets for cancer immunotherapy. Collectively, these data support the ongoing development of IL-17B neutralizing antibodies. Citation Format: Emilie Laprevotte, Aurelie Docquier, Jeremy Bastid, Cecile Dejou, Marion Lapierre, Gilles Alberici, Armand Bensussan, Jean-Francois Eliaou, Nathalie Bonnefoy. Generation of anti-IL-17B antibodies neutralizing IL-17B-mediated alterations of the immune microenvironment, promotion of tumor cell initiating capacity and chemoresistance [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 1602. doi:10.1158/1538-7445.AM2017-1602

Abstract 3222: Preclinical development of a humanized blocking antibody targeting the CD39 immune checkpoint for cancer immunotherapy

CD39 (ENTPD1) is a cell membrane ectonucleotidase that hydrolyzes extracellular immunoactivating ATP and ADP into AMP, which can be further hydrolyzed by ectonucleotidase CD73 into immunosuppressive adenosine. Within the tumor microenvironment, adenosine accumulation causes immune suppression and dysregulation of immune cell infiltrates resulting in tumor spreading. The role of CD39 expression on both Tregs and on tumor cells in promoting immunosuppression has been demonstrated in several reports. Blockade of CD39 may promote anti-tumor immunity by directly accumulating immunostimulating ATP and indirectly by reducing adenosine accumulation. Here, we describe the discovery and preclinical development of an anti-huCD39 blocking antibody for cancer immunotherapy. Parental anti-huCD39 mouse monoclonal antibody was humanized. The humanized mAb specifically binds huCD39 protein, but not CD39-like proteins. Nanomolar affinities for human CD39 were measured in SPR studies on recombinant CD39 protein and in flow cytometry titration studies on CD39 expressing transfectants and tumor cell lines. The humanized mAb blocked human CD39 ATPase activity in vitro in the nanomolar range, as demonstrated using transfected cells, CD39-expressing tumor cell lines, as well as human PBMC and ex-vivo isolated fresh tumor samples. The humanized mAb cross-reacted on cynomolgus CD39 and blocked ATPase activity on cynomolgus PBMC with similar efficacy as on human PBMC. Finally, treatment with blocking anti-CD39 mAb inhibited tumor growth in vivo in mouse tumor models. Taken together, these data support the clinical development of anti-CD39 neutralizing mAb for cancer immunotherapy. Citation Format: Severine Augier, Ivan Perrot, Cecile Dejou, Rachel Joly, Stephane Delahaye, Helene Rispaud Blanc, Caroline Denis, Laurent Gauthier, Armand Bensussan, Jean-francois Eliaou, Yannis Morel, Nathalie Bonnefoy, Jeremy Bastid, Carine Paturel. Preclinical development of a humanized blocking antibody targeting the CD39 immune checkpoint for cancer immunotherapy. [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 3222.

Reply to PMID 23666743

demonstrated increased proliferation, also similar to B cells in PKC -deficient mice. The development of autoimmunity is interpreted as demonstrating that PKC is crucial in regulating B cell tolerance and preventing self-reactivity in humans. We would like to note that PKC activity is reduced in patients with sporadic non-Mendelian lupus, and that this inactivation in T cells can contribute to the development of lupus-like autoimmunity through effects on T cell DNA methylation (3). DNA methylation is an epigenetic mechanism that silences gene expression by promoting a repressive chromatin configuration that is inaccessible to transcription factors. DNA methylation patterns are established during differentiation and serve to suppress genes that are irrelevant or detrimental to the functions of mature cells. The methylation patterns are then replicated each time a cell divides, by the maintenance methyltransferase DNA methyltransferase 1 (DNMT-1). DNMT-1 levels are low in resting T cells but are up-regulated in stimulated T cells by signals transmitted through the ERK pathway. Inhibiting DNA methylation in dividing T cells, either with direct DNMT inhibitors such as 5-azacytidine or the lupusinducing drug procainamide, or with MEK inhibitors such as PD98059 and U0126, alters gene expression and converts normal antigen-specific CD4 T cells into autoreactive, cytotoxic, and proinflammatory T cells that are sufficient to cause lupus-like autoimmunity in animal models (4,5). The role of decreased T cell ERK pathway signaling in causing lupus-like autoimmunity has been confirmed in a double-transgenic mouse model in which dominant-negative MEK can be induced selectively in T cells by adding doxycycline to the drinking water. In these mice, doxycycline decreases T cell DNMT-1 levels, demethylates DNA, activates genes normally suppressed by DNA methylation, and causes lupus-like autoimmunity (6–8). PKC is a multifunctional signaling molecule that plays a crucial role in ERK pathway signaling in T cells (9). Inhibiting T cell PKC with rottlerin or the lupus-inducing drug hydralazine, or by transfection with a dominant-negative PKC similarly demethylates T cell DNA methylation and alters gene expression (3). CD4 T cells treated with hydralazine are sufficient to cause lupus-like autoimmunity in mice (10). Taken together, these findings indicate that inhibiting ERK pathway signaling, through effects on PKC or downstream signaling molecules, is sufficient to cause a lupus-like disease. Importantly, PKC is catalytically inactivated by oxidative damage in CD4 T cells from patients with active lupus, causing decreased ERK pathway signaling, decreased DNMT-1 levels, and demethylation of the same genes normally silenced by DNA methylation (11). These observations suggest that PKC mutations may contribute to lupus through effects on T cells, and the effects on B cells seen in the patients described by Belot and colleagues as well as in PKC -deficient mice may augment these effects. Dr. Richardson serves on the scientific advisory board of Ignyta. Bruce Richardson, MD, PhD Faith M. Strickland, PhD Amr H. Sawalha, MD Gabriela Gorelik, PhD University of Michigan Ann Arbor, MI

Abstract B3: CD39+ cancer cells mediate immunosuppression reverted by CD39-blocking antibodies.

The CD39 and CD73 ectonucleotidases hydrolyze extracellular ATP and ADP into immunosuppressive adenosine that binds adenosine receptor and inhibits T cell and NK cell responses. It has been demonstrated that CD39+ Tregs are increased in some human cancers and participate to immunosuppression. The importance of CD39+ Tregs in promoting tumor growth and metastasis has been evidenced in several models in vivo (for a review, see Bastid J et al., Oncogene, 2012). Here, we addressed whether CD39 is expressed by tumor cells and whether CD39+ tumor cells mediate suppression through the CD39-adenosine pathway. Immunohistochemical staining of normal and tumor tissues revealed that CD39 is upregulated in several types of human cancer. In cancer specimens, CD39 is expressed by infiltrating lymphocytes, tumor stroma and tumor cells, whereas its expression in normal samples is absent or weak and mostly limited to vascular endothelia. The expression of CD39 at the cell surface of tumor cells was further directly demonstrated by flow cytometry in human cancer cell lines. We evidenced that CD39+ tumor cells express functional CD39 and inhibit CD4 and CD8 T cell function in a CD39-dependant manner. Treatment with CD39-blocking antibodies was able to alleviate CD39+ tumor cell-mediated inhibition of CD4 and CD8 T cells in co-culture experiments. In conclusion, interfering with the CD39-adenosine pathway could represent a novel immunotherapy strategy for inhibiting Treg and tumor cell-mediated immunosuppression. Furthermore, these results support the ongoing development of CD39-blocking monoclonal antibodies as potential anticancer drugs. Citation Format: Anne Regairaz, Caroline Laheurte, Nathalie Bonnefoy, Jean-Francois Eliaou, Gilles Alberici, Armand Bensussan, Jeremy Bastid. CD39+ cancer cells mediate immunosuppression reverted by CD39-blocking antibodies. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B3.