Les leucémies aigues myéloïdes (LAM) sont des hémopathies malignes de mauvais pronostic dont les thérapies actuelles ne permettent d’obtenir des taux de survie à 5 ans chez les adultes que d’environ 40%. Par conséquent, il est nécessaire d’approfondir nos connaissances concernant les mécanismes d’oncogenèse pour développer de nouvelles approches thérapeutiques. Malgré leur hétérogénéité clinique et biologique, les LAM ont certaines caractéristiques communes comme l’activation de la voie de signalisation mTORCl qui est détectée dans la plupart des échantillons de LAM. MTORCl contrôle la survie, la croissance et la prolifération cellulaire, notamment via le contrôle de la traduction des ARNm et donc de la synthèse protéique. Au cours de ce travail, nous montrons qu’il existe, dans les LAM, une dérégulation de mTORCl qui explique les limites des effets anti-leucémiques observés avec la rapamycine (un inhibiteur allostérique de mTORCl) et qui est médiée en partie par l’activité de la sérine thréonine kinase Pim2, qui contrôle la phosphorylation de la cible de mTORCl, la protéine 4E-BP1. Cependant, cibler directement la traduction produit des effets anti-leucémiques importants, ce que nous avons montré en utilisant une molécule inhibant spécifiquement le complexe d’initiation de la traduction, le 4EGI-l. EIF4E est essentiel à l’initiation de la traduction et nous avons montré sa surexpression au niveau protéique dans la plupart des échantillons de LAM au diagnostic par comparaison à des cellules hématopoïétiques normales CD34+. Bien que son niveau d’expression n’ait pas de valeur pronostique intrinsèque, ce résultat suggère un potentiel important au blocage de la traduction dans la plupart des cas de LAM. Dans la perspective d’inhiber mTORCl, nous avons activé la voie LKBl/AMPK par la metformine, ce qui a induit des effets anti-leucémiques in vitro et in vivo via une modification du métabolisme cellulaire avec en particulier une inhibition de la synthèse de protéines oncogéniques. La metformine n’étant pas un candidat en thérapeutique dans les LAM du fait d’un index thérapeutique trop étroit, de nouvelles molécules modulant la voie LKBl/AMPK sont en cours de développement. Enfin, nous avons étudié le rôle de la sérine thréonine kinase Pim2, qui contrôle la traduction protéique et la survie dans les cellules de LAM Flt3-ITD+. Nous avons de plus montré que la sur-expression de Pim2 constitue un nouveau mécanisme de résistance aux inhibiteurs de Flt3 et représente donc une cible thérapeutique prometteuse dans cette catégorie de LAM. L’étude de la voie mTORCl et de la traduction permet donc d’envisager de multiples perspectives thérapeutiques dans les LAM dont certaines sont déjà en cours de développement clinique. / Acute myeloid leukemia (AML) are hematological malignancies with adverse prognosis in which therapies only gives 40% survival within 5 years in adults. Hence, it is important to increase our knowledge regarding oncogenesis to further develop new therapeutic approaches. Despite their clinical and biological heterogeneity, AML have in common the constitutive activation of mTORC1 signaling which is detected in most AML samples. MTORC1 controls cell survival, growth and proliferation, in particular through control of mRNA translation and protein synthesis. During this work, we show, in AML, that mTORC1 is deregulated which explain the poor effects observed with rapamycin (a mTORC1 allosteric inhibitor) and is partially mediated by the serine/threonine kinase Pim-2 which controls the mTORC1 target 4E-BP1. Nevertheless, directly targeting translation, using a specific translation initiation inhibitor named 4EGI-1, have important anti leukemic effects. EIF4E is described as essential in translation initiation and we show its protein overexpression in most AML samples at diagnosis compared with normal hematopoietic CD34+ cells. Whereas eIF4E level expression has no prognostic impact, this result suggests an important potential for treatment targeting translation initiation in AML. In our purpose of inhibiting mTORC1, we were able to activate LKB1/AMPK signaling pathway with metformin, which induces anti leukemic effects in vitro and in vivo through in particular oncogenic protein translation inhibition. Metformin is not a good AML therapeutic candidate because of a narrow therapeutic index, new compound targeting LKB1/AMPK are in development. Finally, we studied the role of the serine/threonine kinase Pim-2 and show that it controls protein translation and FLT3-ITD+ AML cells survival. Furthermore, we show that Pim-2 overexpression is a new mechanism of Flt3 inhibitors resistance and represent a new promising therapeutic target in this AML subtype. Overall, mTORC1 and protein translation study in AML show multiple therapeutics perspective, some of them are already in clinical development.

La sénescence est un mécanisme de défense antiprolifératif dont la cellule est munie afin de prévenir l’accumulation de mutations pouvant mener à sa transformation et l’éventuel développement d’une tumeur. Ce programme consiste en un arrêt permanent du cycle cellulaire. Il peut être activé par de nombreux stimuli tels que le raccourcissement des télomères, le stress oxydatif, ou l’expression d’un oncogène constitutivement actif. Sa régulation requiert l’activation de protéines appelées des suppresseurs de tumeur dont les plus importants sont p53 et RB. De manière plus spécifique, les sénescences induites par l’expression des oncogène RASV12 ou STAT5A(1*6) sont respectivement caractérisées par l’augmentation de l’expression des protéines PML et CHES1/FOXN3. Le but de cette thèse est, dans un premier temps, de mettre en évidence le mécanisme de régulation de la sénescence par PML. PML est un suppresseur de tumeur dont l’expression dans des cellules diploïdes normales est suffisante pour induire la sénescence. Cette protéine forme des corps nucléaires sphériques au sein desquels est recruté, parmi d’autres molécules, la protéine du rétinoblastome RB. RB est un régulateur négatif du cycle cellulaire capable de lier et inhiber les facteurs de transcription E2F dont les gènes cibles sont nécessaires à la prolifération. Nos travaux démontrent que le mécanisme d’induction de la sénescence par PML implique le recrutement du complexe RB/E2F aux corps de PML afin de renforcer l’inhibition de l’activité des E2F par RB. Également, les E2F sont recrutés aux corps de PML en compagnie de leurs promoteurs ce qui favorise la formation d’hétérochromatine au niveau de ces gènes, aidant à leur répression et donc à l’établissement de la sénescence. D’autre part, cette thèse a pour but de caractériser le rôle de CHES1/FOXN3 dans la régulation du cycle cellulaire. CHES1 est un facteur de transcription de la famille des Forkheads. Son expression dans des cellules cancéreuses provoque un ralentissement de leur prolifération. Afin de comprendre son mécanisme de fonctionnement, une analyse sur micropuce d’ADN de l’expression des gènes de cellules cancéreuses exprimant CHES1 a été réalisée. Cette analyse a montré que, dans la cellule, CHES1 joue un rôle de répresseur transcriptionnel. Plus précisément, CHES1 réprime, entre autres, l’expression de gènes nécessaires à la synthèse des protéines tels que PIM2 et DYRK3. De manière intéressante, la réexpression de PIM2 dans des cellules cancéreuses exprimant CHES1 permet de rétablir partiellement la prolifération cellulaire. Également, l’analyse sur micropuce a révélé que CHES1 régule l’expression de nombreux gènes impliqués dans la formation des cilia dont l’une des fonctions semble être de moduler la synthèse protéique. Pris ensemble, ces résultats suggèrent que le mécanisme antitumoral de CHES1 consiste en une inhibition de la synthèse de protéines. / Senescence is an antiproliferative defense mechanism that protects the cell against the accumulation of mutations and, eventually, her transformation and the development of a tumor. This program consists of a permanent cell cycle arrest. It can be activated by many stimuli, such as telomere shortening, oxidative stress, or the expression of a constitutively active oncogene. Senescence regulation requires activation of proteins called tumor suppressors which the most important are p53 and RB. More specifically, senescences induced by the expression of oncogenic RASV12 or STAT5A (1 * 6) are respectively characterized by increased expression of the proteins PML and CHES1/FOXN3. The purpose of this thesis is, firstly, to identify the mechanism of regulation of senescence by PML. PML is a tumor suppressor whose expression in normal diploid cells is sufficient to induce senescence. This protein forms spherical nuclear bodies in which is recruited, among other molecules, the retinoblastoma protein RB. RB is a negative regulator of the cell cycle due to its capacity to bind and inhibit the E2F transcription factors whose target genes are necessary for proliferation. Our work demonstrates that the mechanism of induction of senescence by PML involves the recruitment of the complex RB/E2F to the PML body to enhance the inhibition of E2F activity by RB. Also, the E2Fs are recruited to PML bodies together with their promoters which promotes the formation of heterochromatin in these genes, helping their repression and thus the establishment of senescence. On the other hand, this thesis aims to characterize the role of CHES1/FOXN3 in regulating the cell cycle. CHES1 is a transcription factor of the Forkheads family which expression in cancer cells causes a growth reduction. To understand the antitumoral mechanism of CHES1, a microarray analysis of cancer cells expressing CHES1 was performed. This analysis shows that CHES1 is a transcriptional repressor. Specifically, CHES1 represses, among others, the expression of genes required for the synthesis of proteins such as PIM 2 and DYRK3. Interestingly, re-expression of PIM 2 in cancer cells expressing CHES1 partially restores cell proliferation. Also, microarray analysis revealed that CHES1 regulates the expression of many genes involved in the formation of cilia which one of the functions seems to be to modulate protein synthesis. Taken together, these results suggest that the antitumor mechanism of CHES1 involves inhibition of protein synthesis.

One key to the pathogenic potential of the mycobacteria lies in their capacity to resist destruction by infected macrophages and dendritic cells. Robust host immune responses during mycobacterial infection often involve a potent CD4, CD8 and gamma delta T cell mediated effector responses including lysis of mycobacteria infected host cells, secretion of variety of cytokines like IFN-γ etc. However, pathogenic mycobacteria survives for prolonged periods in the phagasomes of infected macrophages within the host in an asymptomatic, latent state and can reactivate years later if the host’s immune system wanes. One of the most devastating consequences of infection with mycobactreia is the formation of caseating granulomas followed by tissue destruction with liquefaction causing cavity formation. Pathogenic mycobacteria reside in these granulomas, which are formed by the accumulation of monocytes, epithelioid and foamy macrophages as well as cytolytic lymphocytes including CD8 T cells around the infection focus. In this regard, rigid balance as well as modulation of inflammatory immune responses by the host upon infection of pathogenic microbes is one of the crucial steps not only in controlling the spread of pathogen from the site of infection to reminder of host organs, but also in mounting an effective memory response so that future exposures/infections by similar pathogen can be effectively controlled. Significantly, despite this complex host response, it remains unclear, that why the immune response controls mycobacteria but does not eradicate infection. Both human and mouse studies have provided ample evidence that even in the face of an adequate immune response, mycobacteria are able to persist inside macrophages. These findings have suggested series of survival strategies employed by Mycobacterium sp. during its infection of host macrophages/dendritic cells which include, blockade of phagosome-lysosome fusion, secretion of ROI antagonistic proteins like superoxide dismutase & catalase, inhibition of processing of its antigens for presentation to T cells, decrease in secretion of proinflammatory cytokines by inducing secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. In view of above-mentioned observations, graulomas in response to pathogenic mycobacterial infections have long been considered host-protective structures formed to contain infection. In this perspective, Matrix metalloproteinase-9 (MMP-9), an important member of Zn2+ and Ca2+ dependent endopeptidases, participates in a significant manner in several aspects of host immune responses to mycobacterial infection such as graunloma formation, matrix (ECM) reorganization, lymphocytes trafficking and infiltrations, inflammation etc. MMP-9 is expressed at various clinical categories of tuberculosis disease like active cavitary tuberculosis, meningitis and pleuritis. Notably, in case of pulmonary tuberculosis, breakdown of ECM by MMP-9 forms an integral part of the granuloma formation. Importantly, Mycobacterium tuberculosis infection in MMP-9 deficient mice revealed defective bacterial proliferation, reduced bacterial burden and reduced lung macrophages recruitment compared to wild-type, in addition, to reduced ability to initiate or maintain well-formed granulomas. In this context, we explored the signaling events modulated by Mycobacterium bovis bacillus Calmette-Gue´rin (BCG) or its novel cell wall antigens during induced expression of MMP-9 or SPI6 in macrophages. Our studies clearly demonstrate that NO, a product of iNOS activity, is responsible for M. bovis BCG-triggered activation of Notch1 in macrophages through direct regulation of Jagged1 expression as well as in generation of activated Notch1. We present the evidence that iNOS activity is a critical factor in TLR2 mediated Notch1 activation as macrophages derived from iNOS knockout (iNOS-/-), but not from wild-type (WT) mice failed to activate Jagged1 expression as well as Notch1 signaling upon M. bovis BCG infection. The loss of TLR2-mediated Jagged1 expression or Notch1 activation in iNOS-/-macrophages could be rescued by treatment with NO donor 3-morpholinosydnonimine (SIN1) or S-nitroso-Nacetylpenicillamine (SNAP). Signaling perturbations strongly implicated the role for cross talk among members of Notch1-PI3 Kinase and MAPK cascades in M. bovis BCG-TLR2– mediated activation of Notch1 target genes MMP-9 or Hes1. Chromatin immunoprecipitation experiments demonstrate that M. bovis BCG’s ability to trigger increased binding of CSL/RBP-Jk to MMP-9 promoter was severely compromised in macrophages derived from iNOS-/-mice compared to WT mice. These results are consistent with the observation that NO-triggered Notch1 signaling-mediated CSL/RBP-Jk recruitment has a positive regulatory role in M. bovis BCG-induced MMP-9 transcription. We show the correlative evidence that this mechanism operates in vivo by immunohistochemical expression analysis of activated Notch1 or its target gene products Hes1 or MMP-9 in brains of WT or iNOS-/-mice that were intracerebrally infected with M. bovis BCG. Further, activation of Notch1 signaling in vivo could be demonstrated only in granulomatous lesions in brains derived from human patients with tuberculous meningitis (TBM) as opposed to healthy individuals, validating the role of Notch1 signaling in mycobacterial pathogenesis. Briefly, we have identified NO as the pathological link between TLR2 and Notch1 signaling, which regulates the relative abundance of various immunopathological parameters including MMP-9 in macrophages. Synopsis Despite mycobacteria elicits robust host T cell responses as well as production of NO, ROI or cytokines like interferon-γ (IFN-γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. These findings clearly advocate roles for mycobacteria mediated various immune evasion strategies to modulate the signaling cascades thus leading to macrophage activation. Importantly, TLR2 triggering by mycobacteria elicits the activation of divers sets of anti or pro-apototic genes expression, a balance of which will have strong bearing on the overall cell-fate decisions across many cell types. In this regard, a novel granzyme B inhibitor, SPI6/PI9, can exhibit robust resistance to various cells including dendritic cells or tumor cells from lysis by CD8 cytotoxic T cells (CTL). SPI6/PI9 predominantly functions by inhibiting Granzyme B, an effector protease of cytotoxic granules released by CTL upon its TCR recognition of infected cells such as macrophages, dendritic cells etc. In this context, current investigation attempted to investigate molecular details involved in M. bovis BCG triggered SPI6 expression as well as the involvement of TLR2NO-Notch1 signaling axis in driving induced expression of SPI6, akin to that of MMP-9 expression. We demonstrate that M. bovis BCG trigger SPI6 expression in macrophages and requires critical participation of TLR2-MyD88 dependent NO-Notch1 signaling events. More importantly, signaling perturbations data suggest the involvement of cross talk among the members of PI3 Kinase and MAPK cascades with Notch1 signaling in SPI6 expression. In addition, SPI6 expression requires the Notch1 mediated recruitment of CSL/RBP-Jk and NF-κB to the SPI6 promoter. Functional studies strongly attribute critical involvement of SPI6 and MMP-9 in imparting protection to M.bovis BCG infected macrophages from lysis effectuated by CTL. Macrophages are principal mediators of initiation as well as activation of host inflammatory responses to pathogenic mycobacterial infection. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, envelope glycoconjugates like Lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), Trehalose 6,6′dimycolate (TDM; cord factor) etc. are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. A number of biological functions have been credited to PIM2. PIM2 was shown to trigger TLR2 mediated activation of macrophages that resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. In addition to pulmonary granuloma-forming activities, PIM2 was shown to recruit NKT cells into granulomas. Further, surface associated PIM was suggested to act as adhesins mediating attachment of M. tuberculosis bacilli to non-phagocytic cells. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether novel cell surface antigen PIM2 similar to whole M. bovis BCG bacilli can contribute to molecular signaling events leading to MMP-9 expression in macrophages. Our current study provides the evidence that PIM2 driven activation of signaling cascades triggers the expression of MMP-9. TLR stimulation by various agonists has been shown to activate Notch signaling resulting in modulation of diverse target genes involved in pro-inflammatory responses in macrophages. In this regard we demonstrated that PIM2 induced expression of MMP-9 involved Notch1 upregulation and activation of Notch1 signaling pathway in a TLR2-MyD88 manner. Enforced expression of the cleaved Notch1 in macrophages induced the expression of MMP-9. Further, PIM2 triggered significant p65 nuclear factor-κB (NF-κB) nuclear translocation that was dependent on activation of PI3 Kinase or Notch1 signaling. Furthermore, MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NFκB to MMP-9 promoter. Taken together, our observations clearly describe involvement of TLR2/iNOS in activating Notch1 and PI3 Kinase signaling during infection of macrophages with M. bovis BCG, thus effectuating the regulation of specific effector gene expressions, such as SPI6 and MMP-9. These results clearly describe the cross talk of Notch1 signaling with PI3 Kinase and MAPK pathways, thus leading to differential effects of Notch1 signaling. Overall, we believe that our work will extend the current understanding of inflammatory parameters associated with host-mycobacteria interactions which might lead to better design as well as evaluation of therapeutic potential of novel agents targeted at diverse mycobacterial diseases.

Liver cancer is the fourth leading cause of cancer-related mortality worldwide with limited therapeutic options. Thus, novel treatment strategies are urgently required. While the oncogenic kinase, proviral integration site for Moloney murine leukemia virus 2 (PIM2), has been shown to be overexpressed in liver cancer, little is known about the role of PIM2 in this tumor entity. In this study, we explored the functional relevance and therapeutic potential of PIM2 in liver cancer. Using PIM2‐specific siRNAs, we examined the effects of PIM2 knockdown on proliferation (WST-1 assays and spheroid assays), 3D-colony formation and colony spread, apoptosis (flow cytometry and caspase 3/caspase 7 activity), as well as cell cycle progression (flow cytometry, RT-qPCR and western blot analysis) in the two liver cancer cell lines, HepG2 and Huh‐7. In subcutaneous liver cancer xenografts, we assessed the effects of PIM2 knockdown on tumor growth via the systemic delivery of polyethylenimine (PEI)-complexed siRNA. The knockdown of PIM2 resulted in potent anti-proliferative effects in cells grown on plastic dishes, as well as in spheroids. This was due to G0/G1 cell cycle blockade and the subsequent downregulation of genes related to the S phase as well as the G2/M phase of the cell cycle, whereas the apoptotic rates remained unaltered. Furthermore, colony formation and colony spread were markedly inhibited by PIM2 knockdown. Notably, we found that HepG2 cells were more sensitive to PIM2 knockdown than the Huh‐7 cells. In vivo, the therapeutic nanoparticle-mediated delivery of PIM2 siRNA led to profound anti-tumor effects in a liver cancer xenograft mouse model. On the whole, the findings of this study underscore the oncogenic role of PIM2 and emphasize the potential of targeted therapies based on the specific inhibition of PIM2 in liver cancer.

Pathogenic Mycobacteria are among the most unrelenting pathogens known to mankind as one-third of the world population is latently infected with Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis. Despite many species of mycobacteria elicits robust host T cell responses as well as production of cytokines like interferon-γ (IFN- γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. One potential mechanism by which mycobacteria may achieve a state of long-term persistence amid a robust host immune response is by modulating the signaling cascades leading to macrophage activation. Activation of proinflammatory responses by the host macrophages upon infection with mycobacteria requires the involvement of a variety of signaling events. Studies have indicated that macrophages infected with pathogenic mycobacteria produce significantly less tumor necrosis factor (TNF)-α and other proinflammatory molecules compared with infection with nonpathogenic mycobacteria, which likely play a role in enhancing mycobacterial survival in vivo. Furthermore, macrophages infected with mycobacteria become refractory to many cytokines including IFN-γ and modulation of host cell signaling responses is critical for the suppression of a generalized inflammatory response which might influence the persistence of mycobacteria within the host. In this context, Suppressor of cytokine signaling (SOCS) 3, a member of SOCS family function as negative regulators of multiple cytokine and toll like receptor induced signaling. The SOCS3 has been shown to specifically inhibit signaling by IFN-γ, IL-6 family of cytokines and can act as a negative regulator of inflammatory responses. In this regard, many species of mycobacteria including M. bovis BCG triggers the inducible expression of SOCS3. Further, it has been suggested that M. bovis BCG triggered SOCS3 and SOCS1 proteins leads to the inhibition of IFN- γ stimulated JAK/STAT signaling in macrophages. Albeit JAK/STAT signaling pathway is generally believed to be involved, STAT-independent signals are suggested to take part in the induction of SOCS proteins in many systems signifying the involvement of multiple signal pathways in regulation of SOCS expression. Further little is known about the early, receptor proximal signaling mechanisms underlying mycobacteria-mediated induction of SOCS3. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, many cell wall antigens like LAM, PIM, TDM, PE family antigens etc are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extra cellular environment in the form of exocytosed vesicles. In this context, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. PIM are suggested to be the common anchor of LM and LAM as PIM, LM, and LAM originate from identical biosynthetic pathway. PIM are present in virulent M. tuberculosis H37Rv as well as in M. bovis BCG and a number of biological functions have been recently credited to PIM2. PIM2 is suggested to trigger the activation of cells via Toll like receptor (TLR)-2 and stimulation resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. PIM2 induces proinflammatory stimuli such as TNF-α and IL-12 in murine and human macrophages in a TLR2 dependent manner. PIM exhibited pulmonary granuloma-forming activities as well as was shown to be responsible for the recruitment of NKT cells to granulomas. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether M. bovis BCG or novel cell surface antigens like PIM2 or Rv0978c, a PE-PGRS protein with unknown function can contribute to M. bovis BCG triggered molecular signaling events leading to SOCS3 expression in macrophages. Our studies clearly demonstrated that M. bovis BCG can trigger SOCS3 expression in macrophages. The inception of signaling by M. bovis BCG is TLR2-MyD88 dependent, but not TLR4 dependent. The perturbation of TLR2 signaling and the downregulation of MyD88 resulted in significant decrease in SOCS3 expression implicating the role of TLR2-MyD88 axis in M. bovis BCG triggered signaling. Experiments with cycloheximide and neutralizing antibodies to IL-10 evinced that M. bovis BCG triggered SOCS3 expression is a primary response and requires direct activation of signaling cascades. In the current study, we show for the first time that infection of macrophages with M. bovis BCG activates NOTCH1 signaling events, which leads to expression of SOCS3. The perturbation of NOTCH signaling in infected macrophages either by siRNA mediated down regulation of NOTCH1 or RBP-Jk or by inhibition with pharmacological inhibitor gamma secretase-I, resulted in the marked reduction in the expression of SOCS3. Further, the enforced expression of the NOTCH1 intracellular domain (NICD) in RAW264.7 macrophages induces the expression of SOCS3, which can be further potentiated by M. bovis BCG. Furthermore, the inhibition of TLR2 signaling by a TLR2 dominant-negative construct resulted in inhibition of NOTCH1 activation. Additionally, our results demonstrates for the first time that physical association of TLR2 with both Phosphoinositide-3 Kinase (PI3K) and NOTCH1, which suggest the significant role of TLR2 triggering by of M. bovis BCG in the activation of PI3K and NOTCH1. More importantly, signaling perturbations data suggest the involvement of cross-talk among the members of PI3K and MAPK cascades with NOTCH1 signaling in SOCS3 expression. In addition, SOCS3 expression requires the NOTCH1 mediated recruitment of CSL/RBP-Jk and Nuclear Factor-B (NF-B) to the SOCS3 promoter. A number of biological functions triggered by mycobacteria are often attributed to many of the cell wall antigens. As part of our current investigation, we explored whether two novel cell wall associated antigens namely PIM2 and a PE-PGRS antigen, Rv0978c could play as significant or crucial cell wall ingredients which imparts ability to M. bovis BCG to trigger activation of NOTCH signaling leading to SOCS3 expression. Akin to M. bovis BCG, PIM2 activates NOTCH1 signaling resulting NICD formation which leads to the expression of SOCS3 in a TLR2-MyD88 dependent manner. PIM2 mediated NOTCH1 activation, both directly influences the SOCS3 expression by serving as coactivator in RBP-Jk complex and indirectly triggers SOCS3 expression by activating PI3K-MAPK-NF-κB cascade. One important outcome of the genome sequencing project of M. tuberculosis was the discovery of two new multigene families designated PE and PPE, named for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. Many PE and PPE proteins are composed only of PE or PPE homologous domains. However, in other proteins, the PE domain is often linked to a unique domain of various lengths that is rich in alanine and glycine amino acids, termed the PGRS domain (PE-PGRS subfamily). PE family genes were suggested to play roles in the virulence of the pathogen and many members of PE family proteins are reported be localized on the surface of M. tuberculosis bacilli. Some of the PE proteins may play a role in immune evasion and antigenic variation or may be linked to virulence. Additionally, it has been suggested that the PE-PGRS subfamily of PE genes is enriched in genes with a high probability of being essential for M. tuberculosis. The uniqueness of the PE genes is further illustrated by the fact that these genes are restricted to mycobacteria. However, despite their abundance in mycobacteria, very little is known regarding the expression or the functions of PE family genes. In this context, we have chosen to study Rv0978c as a typical member of PE-PGRS family based on the following observations. Rv0978c was upregulated in TB bacilli upon infection of macrophages. Rv0978c was demonstrated to be a member of a group of genes called in vivo-expressed genomic island, which were shown to be upregulated in M. tuberculosis bacilli during infection of mice. Rv0978c was also shown to be upregulated, at least eightfold, in human brain microvascular endothelial cell-associated M. tuberculosis infection, suggesting a role for endothelial cell invasion and intracellular survival. In the current investigation, we have demonstrated that Rv0978c is hypoxia responsive gene based on promoter analysis and upregulated in M. tuberculosis during the infection of macrophages. Further, Rv0978c is associated with cell wall and is exposed outside the surface of the bacterium suggesting the possible access to intracellular compartments of the infected macrophages. In this perspective, our results clearly demonstrate that Rv0978c triggers SOCS3 expression by activating PI3K-ERK1/2-NF-B cascade in mouse macrophages. Additionally, Rv0978c elicited humoral antibody reactivities in a panel of human sera or in cerebrospinal fluid samples obtained from different clinical categories of tuberculosis patients. DNA immunizations experiments in mice clearly suggested that Rv0978c is an immunodominant antigen demonstrating significant T cell and humoral reactivites. These observations clearly advocate that Rv0978c protein is expressed in vivo during active infection with M. tuberculosis and that the Rv0978c is immunogenic. These results clearly describe the cross-talk of NOTCH1 signaling with signaling pathways like PI3K and MAPK pathways during infection of macrophages with M. bovis BCG eventually resulting in regulation of specific gene expressions, such as SOCS3. These observations lead to a possibility of differential effects of NOTCH1 signaling activated upon infection by an intracellular bacillus, which could be involved in modulation of macrophage functions depending on a local immunological milieu. Taken together, our findings suggest that, induction of Suppressors of Cytokine Signaling 3 molecule by M. bovis BCG or by its cell wall antigens represents a crucial immune subversion mechanism in order to suppress or attenuate host responses to cytokines to generate the conditions that favor survival of the mycobacteria.